Polypeptides having beta-glucosidase activity and polynucleotides encoding same

ABSTRACT

The present invention relates to isolated polypeptides having beta-glucosidase activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 13/328,893, filed Dec. 16, 2011, which is a divisional applicationof U.S. application Ser. No. 11/997,625, filed Feb. 1, 2008, now U.S.Pat. No. 8,097,772, which is a 35 U.S.C. 371 national application ofPCT/US2006/030719 filed on Aug. 4, 2006 and claims priority from U.S.Provisional Application Ser. No. 60/705,607 filed on Aug. 4, 2005, whichapplications are fully incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to isolated polypeptides havingbeta-glucosidase activity and isolated polynucleotides encoding thepolypeptides. The invention also relates to nucleic acid constructs,vectors, and host cells comprising the polynucleotides as well asmethods for producing and using the polypeptides.

2. Description of the Related Art

Cellulose is a polymer of the simple sugar glucose covalently bonded bybeta-1,4-linkages. Many microorganisms produce enzymes that hydrolyzebeta-linked glucans. These enzymes include endoglucanases,cellobiohydrolases, and beta-glucosidases. Endoglucanases digest thecellulose polymer at random locations, opening it to attack bycellobiohydrolases. Cellobiohydrolases sequentially release molecules ofcellobiose from the ends of the cellulose polymer. Cellobiose is awater-soluble beta-1,4-linked dimer of glucose. Beta-glucosidaseshydrolyze cellobiose to glucose.

The conversion of cellulosic feedstocks into ethanol has the advantagesof the ready availability of large amounts of feedstock, thedesirability of avoiding burning or land filling the materials, and thecleanliness of the ethanol fuel. Wood, agricultural residues, herbaceouscrops, and municipal solid wastes have been considered as feedstocks forethanol production. These materials primarily consist of cellulose,hemicellulose, and lignin. Once the cellulose is converted to glucose,the glucose is easily fermented by yeast into ethanol. Since glucose isreadily fermented to ethanol by a variety of yeasts while cellobiose isnot, any cellobiose remaining at the end of the hydrolysis represents aloss of yield of ethanol. More importantly, cellobiose is a potentinhibitor of endoglucanases and cellobiohydrolases. The accumulation ofcellobiose during hydrolysis is undesirable for ethanol production.

Cellobiose accumulation has been a major problem in enzymatic hydrolysisbecause cellulase-producing microorganisms may produce littlebeta-glucosidase. The low amount of beta-glucosidase results in ashortage of capacity to hydrolyze the cellobiose to glucose. Severalapproaches have been used to increase the amount of beta-glucosidase incellulose conversion to glucose.

One approach is to produce beta-glucosidase using microorganisms thatproduce little cellulase, and add the beta-glucosidase exogenously toendoglucanase and cellobiohydrolase to enhance the hydrolysis. However,the quantities required are too costly for a commercial biomass toethanol operation.

A second approach is to carry out cellulose hydrolysis simultaneouslywith fermentation of the glucose by yeast. This process is known assimultaneous saccharification and fermentation (SSF). In an SSF system,fermentation of the glucose removes it from solution. However, SSFsystems are not yet commercially viable because the operatingtemperature for yeast of 28° C. is too low for the 50° C. conditionsrequired.

A third approach to overcome the shortage of beta-glucosidase is tooverexpress the beta-glucosidase in a host, thereby increasing the yieldof beta-glucosidase.

It would be an advantage in the art to provide new beta-glucosidaseswith improved properties for converting cellulosic materials tomonosaccharides, disaccharides, and polysaccharides.

It is an object of the present invention to provide new polypeptideshaving beta-glucosidase activity and polynucleotides encoding thepolypeptides.

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides havingbeta-glucosidase activity selected from the group consisting of:

(a) a polypeptide comprising an amino acid sequence which has at least70% identity with the mature polypeptide of SEQ ID NO: 2;

(b) a polypeptide which is encoded by a polynucleotide which hybridizesunder at least medium stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNA sequencecontained in the mature polypeptide coding sequence of SEQ ID NO: 1, or(iii) a complementary strand of (i) or (ii);

(c) a polypeptide comprisingA-E-[ST][IV][KR]G-[IM]-Q-[DS]-[ST]-G-V-[IV]-A; and

(d) a variant comprising a conservative substitution, deletion, and/orinsertion of one or more amino acids of the mature polypeptide of SEQ IDNO: 2.

The present invention also relates to isolated polynucleotides encodingpolypeptides having beta-glucosidase activity, selected from the groupconsisting of:

(a) a polynucleotide encoding a polypeptide comprising an amino acidsequence which has at least 70% identity with the mature polypeptide ofSEQ ID NO: 2;

(b) a polynucleotide having at least 70% identity with the maturepolypeptide coding sequence of SEQ ID NO: 1; and

(c) a polynucleotide which hybridizes under at least medium stringencyconditions with (i) the mature polypeptide coding sequence of SEQ ID NO:1, (ii) the cDNA sequence contained in the mature polypeptide codingsequence of SEQ ID NO: 1, or (iii) a complementary strand of (i) or(ii); and

(d) a polynucleotide encoding a polypeptide having beta-glucosidaseactivity, wherein the polypeptide comprisesA-E-[ST][IV]-[KR]-G-[IM]-Q-[DS]-[ST]-G-V-[IV]-A.

In a preferred aspect, the mature polypeptide is amino acids 37 to 878of SEQ ID NO: 2. In another preferred aspect, the mature polypeptidecoding sequence is nucleotides 171 to 2753 of SEQ ID NO: 1.

The present invention also relates to nucleic acid constructs,recombinant expression vectors, recombinant host cells comprising thepolynucleotides, and methods of producing the polypeptides havingbeta-glucosidase activity.

The present invention also relates to a plants comprising the isolatedpolynucleotides encoding the polypeptides having beta-glucosidaseactivity.

The present invention also relates to methods for using the polypeptideshaving beta-glucosidase activity in the conversion of cellulosicmaterial to glucose or other substances.

The present invention also relates to detergent compositions comprisingpolypeptides having beta-glucosidase activity.

The present invention also relates to isolated polynucleotides encodinga signal peptide comprising or consisting of amino acids 1 to 19 of SEQID NO: 2, to isolated polynucleotides encoding a propeptide comprisingor consisting of amino acids 20 to 36 of SEQ ID NO: 2, and to isolatedpolynucleotides encoding a prepropeptide comprising or consisting ofamino acids 1 to 36 of SEQ ID NO: 2.

The present invention further relates to nucleic acid constructscomprising a gene encoding a protein, wherein the gene is operablylinked to one or both of a first nucleotide sequence encoding a signalpeptide comprising or consisting of amino acids 1 to 19 of SEQ ID NO: 2and a second nucleotide sequence encoding a propeptide comprising orconsisting of amino acids 20 to 36 of SEQ ID NO: 1, wherein the gene isforeign to the first and second nucleotide sequences.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the genomic DNA sequence and the deduced amino acidsequence of a Penicillium brasilianum strain IBT 20888 beta-glucosidase(SEQ ID NOs: 1 and 2, respectively).

FIG. 2 shows a restriction map of pCR2.1GH3A.

FIG. 3 shows a restriction map of pKKAB.

FIG. 4 shows a restriction map of pKBK01.

FIG. 5 shows the relative activity of the Penicillium brasilianum strainIBT 20888 beta-glucosidase at different pH values as a function oftemperature.

FIG. 6 shows the relative activity of the Penicillium brasilianum strainIBT 20888 beta-glucosidase at different temperatures as a function ofpH.

FIG. 7 shows the residual activity of Novozym 188 after 24 hours ofincubation at different temperatures and pHs.

FIG. 8 shows the residual activity of the Penicillium brasilianum strainIBT 20888 beta-glucosidase after 24 hours of incubation at differenttemperatures and pHs.

FIG. 9 shows the initial reaction rate at different4-nitrophenyl-beta-D-glucopyranose concentrations for the Penicilliumbrasilianum strain IBT 20888 beta-glucosidase.

FIG. 10 shows the initial reaction rate at different cellobioseconcentrations for the Penicillium brasilianum strain IBT 20888beta-glucosidase.

DEFINITIONS

Beta-glucosidase activity: The term “beta-glucosidase” is defined hereinas a beta-D-glucoside glucohydrolase (E.C. 3.2.1.21) which catalyzes thehydrolysis of terminal non-reducing beta-D-glucose residues with therelease of beta-D-glucose. Cellobiase is synonymous withbeta-glucosidase. For purposes of the present invention,beta-glucosidase activity is determined at 25° C. using 1 mM4-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodiumcitrate pH 4.8. One unit of beta-glucosidase activity is defined as 1.0μmole of 4-nitrophenol produced per minute at 25° C., pH 4.8.

The polypeptides of the present invention have at least 20%, preferablyat least 40%, more preferably at least 50%, more preferably at least60%, more preferably at least 70%, more preferably at least 80%, evenmore preferably at least 90%, most preferably at least 95%, and evenmost preferably at least 100% of the beta-glucosidase activity of thepolypeptide consisting of the amino acid sequence shown as amino acids37 to 878 of SEQ ID NO: 2.

Family 3 glycoside hydrolase or family GH3: The term “Family 3 glycosidehydrolase” or “Family GH3” or “Cel3” is defined herein as a polypeptidefalling into the glycoside hydrolase Family 3 according to Henrissat B.,1991, A classification of glycosyl hydrolases based on amino-acidsequence similarities, Biochem. J. 280: 309-316, and Henrissat andBairoch, 1996, Updating the sequence-based classification of glycosylhydrolases, Biochem. J. 316: 695-696.

Isolated polypeptide: The term “isolated polypeptide” as used hereinrefers to a polypeptide which is at least 20% pure, preferably at least40% pure, more preferably at least 60% pure, even more preferably atleast 80% pure, most preferably at least 90% pure, and even mostpreferably at least 95% pure, as determined by SDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation which contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, more preferably atmost 3%, even more preferably at most 2%, most preferably at most 1%,and even most preferably at most 0.5% by weight of other polypeptidematerial with which it is natively or recombinantly associated. It is,therefore, preferred that the substantially pure polypeptide is at least92% pure, preferably at least 94% pure, more preferably at least 95%pure, more preferably at least 96% pure, more preferably at least 96%pure, more preferably at least 97% pure, more preferably at least 98%pure, even more preferably at least 99%, most preferably at least 99.5%pure, and even most preferably 100% pure by weight of the totalpolypeptide material present in the preparation.

The polypeptides of the present invention are preferably in asubstantially pure form. In particular, it is preferred that thepolypeptides are in “essentially pure form”, i.e., that the polypeptidepreparation is essentially free of other polypeptide material with whichit is natively or recombinantly associated. This can be accomplished,for example, by preparing the polypeptide by means of well-knownrecombinant methods or by classical purification methods.

Herein, the term “substantially pure polypeptide” is synonymous with theterms “isolated polypeptide” and “polypeptide in isolated form.”

Mature polypeptide: The term “mature polypeptide” is defined herein as apolypeptide having beta-glucosidase activity that is in its final formfollowing translation and any post-translational modifications, such asN-terminal processing, C-terminal truncation, glycosylation,phosphorylation, etc.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” is defined herein as a nucleotide sequence that encodes amature polypeptide having beta-glucosidase activity.

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined with the FASTA program package,version 3.4 (Pearson and D. J. Lipman, 1988, PNAS 85:2444, and Pearson,1990, Methods in Enzymology 183:63) using default parameters. Thepairwise alignments from the package's Smith-Waterman algorithm(Waterman et al., 1976, Adv. Math. 20: 367) were used for determinationof percent identity. Default parameters included a gap open penalty of−12, a gap extension penalty of −2, and the BLOSUM50 comparison matrix.

For purposes of the present invention, the degree of identity betweentwo nucleotide sequences is determined by the Wilbur-Lipman method(Wilbur and Lipman, 1983, Proceedings of the National Academy of ScienceUSA 80: 726-730) using the LASERGENE™ MEGALIGN™ software (DNASTAR, Inc.,Madison, Wis.) with an identity table and the following multiplealignment parameters: Gap penalty of 10 and gap length penalty of 10.Pairwise alignment parameters are Ktuple=3, gap penalty=3, andwindows=20.

Polypeptide fragment: The term “polypeptide fragment” is defined hereinas a polypeptide having one or more amino acids deleted from the aminoand/or carboxyl terminus of the mature polypeptide of SEQ ID NO: 2 or ahomologous sequence thereof, wherein the fragment has beta-glucosidaseactivity. Preferably, a fragment contains at least 720 amino acidresidues, more preferably at least 760 amino acid residues, and mostpreferably at least 800 amino acid residues.

Subsequence: The term “subsequence” is defined herein as a nucleotidesequence having one or more nucleotides deleted from the 5′ and/or 3′end of SEQ ID NO: 1 or a homologous sequence thereof, wherein thesubsequence encodes a polypeptide fragment having beta-glucosidaseactivity. Preferably, a subsequence contains at least 2160 nucleotides,more preferably at least 2280 nucleotides, and most preferably at least2400 nucleotides.

Allelic variant: The term “allelic variant” denotes herein any of two ormore alternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

Isolated polynucleotide: The term “isolated polynucleotide” as usedherein refers to a polynucleotide which is at least 20% pure, preferablyat least 40% pure, more preferably at least 60% pure, even morepreferably at least 80% pure, most preferably at least 90% pure, andeven most preferably at least 95% pure, as determined by agaroseelectrophoresis.

Substantially pure polynucleotide: The term “substantially purepolynucleotide” as used herein refers to a polynucleotide preparationfree of other extraneous or unwanted nucleotides and in a form suitablefor use within genetically engineered protein production systems. Thus,a substantially pure polynucleotide contains at most 10%, preferably atmost 8%, more preferably at most 6%, more preferably at most 5%, morepreferably at most 4%, more preferably at most 3%, even more preferablyat most 2%, most preferably at most 1%, and even most preferably at most0.5% by weight of other polynucleotide material with which it isnatively or recombinantly associated. A substantially purepolynucleotide may, however, include naturally occurring 5′ and 3′untranslated regions, such as promoters and terminators. It is preferredthat the substantially pure polynucleotide is at least 90% pure,preferably at least 92% pure, more preferably at least 94% pure, morepreferably at least 95% pure, more preferably at least 96% pure, morepreferably at least 97% pure, even more preferably at least 98% pure,most preferably at least 99%, and even most preferably at least 99.5%pure by weight. The polynucleotides of the present invention arepreferably in a substantially pure form. In particular, it is preferredthat the polynucleotides disclosed herein are in “essentially pureform”, i.e., that the polynucleotide preparation is essentially free ofother polynucleotide material with which it is natively or recombinantlyassociated. Herein, the term “substantially pure polynucleotide” issynonymous with the terms “isolated polynucleotide” and “polynucleotidein isolated form.” The polynucleotides may be of genomic, cDNA, RNA,semisynthetic, synthetic origin, or any combinations thereof.

cDNA: The term “cDNA” is defined herein as a DNA molecule which can beprepared by reverse transcription from a mature, spliced, mRNA moleculeobtained from a eukaryotic cell. cDNA lacks intron sequences that areusually present in the corresponding genomic DNA. The initial, primaryRNA transcript is a precursor to mRNA which is processed through aseries of steps before appearing as mature spliced mRNA. These stepsinclude the removal of intron sequences by a process called splicing.cDNA derived from mRNA lacks, therefore, any intron sequences.

Nucleic acid construct: The term “nucleic acid construct” as used hereinrefers to a nucleic acid molecule, either single- or double-stranded,which is isolated from a naturally occurring gene or which is modifiedto contain segments of nucleic acids in a manner that would nototherwise exist in nature. The term nucleic acid construct is synonymouswith the term “expression cassette” when the nucleic acid constructcontains the control sequences required for expression of a codingsequence of the present invention.

Control sequence: The term “control sequences” is defined herein toinclude all components, which are necessary or advantageous for theexpression of a polynucleotide encoding a polypeptide of the presentinvention. Each control sequence may be native or foreign to thenucleotide sequence encoding the polypeptide or native or foreign toeach other. Such control sequences include, but are not limited to, aleader, polyadenylation sequence, propeptide sequence, promoter, signalpeptide sequence, and transcription terminator. At a minimum, thecontrol sequences include a promoter, and transcriptional andtranslational stop signals. The control sequences may be provided withlinkers for the purpose of introducing specific restriction sitesfacilitating ligation of the control sequences with the coding region ofthe nucleotide sequence encoding a polypeptide.

Operably linked: The term “operably linked” denotes herein aconfiguration in which a control sequence is placed at an appropriateposition relative to the coding sequence of the polynucleotide sequencesuch that the control sequence directs the expression of the codingsequence of a polypeptide.

Coding sequence: When used herein the term “coding sequence” means anucleotide sequence, which directly specifies the amino acid sequence ofits protein product. The boundaries of the coding sequence are generallydetermined by an open reading frame, which usually begins with the ATGstart codon or alternative start codons such as GTG and TTG and endswith a stop codon such as TAA, TAG, and TGA. The coding sequence may bea DNA, cDNA, or recombinant nucleotide sequence.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” is defined herein as alinear or circular DNA molecule that comprises a polynucleotide encodinga polypeptide of the invention, and which is operably linked toadditional nucleotides that provide for its expression.

Host cell: The term “host cell”, as used herein, includes any cell typewhich is susceptible to transformation, transfection, transduction, andthe like with a nucleic acid construct or expression vector comprising apolynucleotide of the present invention.

Modification: The term “modification” means herein any chemicalmodification of the polypeptide consisting of the mature polypeptide ofSEQ ID NO: 2; or a homologous sequence thereof; as well as geneticmanipulation of the DNA encoding such a polypeptide. The modificationcan be substitutions, deletions and/or insertions of one or more aminoacids as well as replacements of one or more amino acid side chains.

Artificial variant: When used herein, the term “artificial variant”means a polypeptide having beta-glucosidase activity produced by anorganism expressing a modified nucleotide sequence of the maturepolypeptide coding sequence of SEQ ID NO: 1; or a homologous sequencethereof. The modified nucleotide sequence is obtained through humanintervention by modification of the nucleotide sequence disclosed in SEQID NO: 1; or a homologous sequence thereof.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Beta-Glucosidase Activity

In a first aspect, the present invention relates to isolatedpolypeptides comprising an amino acid sequence which has a degree ofidentity to the mature polypeptide of SEQ ID NO: 2 of at least 60%,preferably at least 65%, more preferably at least 70%, more preferablyat least 75%, more preferably at least 80%, more preferably at least85%, even more preferably at least 90%, most preferably at least 95%,and even most preferably at least 96%, 97%, 98%, or 99%, which havebeta-glucosidase activity (hereinafter “homologous polypeptides”). In apreferred aspect, the homologous polypeptides have an amino acidsequence which differs by ten amino acids, preferably by five aminoacids, more preferably by four amino acids, even more preferably bythree amino acids, most preferably by two amino acids, and even mostpreferably by one amino acid from the mature polypeptide of SEQ ID NO:2.

A polypeptide of the present invention preferably comprises the aminoacid sequence of SEQ ID NO: 2 or an allelic variant thereof; or afragment thereof that has beta-glucosidase activity. In a preferredaspect, a polypeptide comprises the amino acid sequence of SEQ ID NO: 2.In another preferred aspect, a polypeptide comprises the maturepolypeptide of SEQ ID NO: 2. In another preferred aspect, a polypeptidecomprises amino acids 37 to 878 of SEQ ID NO: 2, or an allelic variantthereof; or a fragment thereof that has beta-glucosidase activity. Inanother preferred aspect, a polypeptide comprises amino acids 37 to 878of SEQ ID NO: 2. In another preferred aspect, a polypeptide consists ofthe amino acid sequence of SEQ ID NO: 2 or an allelic variant thereof;or a fragment thereof that has beta-glucosidase activity. In anotherpreferred aspect, a polypeptide consists of the amino acid sequence ofSEQ ID NO: 2. In another preferred aspect, a polypeptide consists of themature polypeptide of SEQ ID NO: 2. In another preferred aspect, apolypeptide consists of amino acids 37 to 878 of SEQ ID NO: 2 or anallelic variant thereof; or a fragment thereof that has beta-glucosidaseactivity. In another preferred aspect, a polypeptide consists of aminoacids 37 to 878 of SEQ ID NO: 2.

In a second aspect, the present invention relates to isolatedpolypeptides having beta-glucosidase activity which are encoded bypolynucleotides which hybridize under at least very low stringencyconditions, preferably at least low stringency conditions, morepreferably at least medium stringency conditions, more preferably atleast medium-high stringency conditions, even more preferably at leasthigh stringency conditions, and most preferably at least very highstringency conditions with (i) the mature polypeptide coding sequence ofSEQ ID NO: 1, (ii) the cDNA sequence contained in the mature polypeptidecoding sequence of SEQ ID NO: 1, (iii) a subsequence of (i) or (ii), or(iv) a complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual,2d edition, Cold Spring Harbor, N.Y.). A subsequence of the maturepolypeptide coding sequence of SEQ ID NO: 1 contains at least 100contiguous nucleotides or preferably at least 200 contiguousnucleotides. Moreover, the subsequence may encode a polypeptide fragmentwhich has beta-glucosidase activity. In a preferred aspect, the maturepolypeptide coding sequence is nucleotides 171 to 2753 of SEQ ID NO: 1.

The nucleotide sequence of SEQ ID NO: 1 or a subsequence thereof, aswell as the amino acid sequence of SEQ ID NO: 2 or a fragment thereof,may be used to design a nucleic acid probe to identify and clone DNAencoding polypeptides having beta-glucosidase activity from strains ofdifferent genera or species according to methods well known in the art.In particular, such probes can be used for hybridization with thegenomic or cDNA of the genus or species of interest, following standardSouthern blotting procedures, in order to identify and isolate thecorresponding gene therein. Such probes can be considerably shorter thanthe entire sequence, but should be at least 14, preferably at least 25,more preferably at least 35, and most preferably at least 70 nucleotidesin length. It is, however, preferred that the nucleic acid probe is atleast 100 nucleotides in length. For example, the nucleic acid probe maybe at least 200 nucleotides, preferably at least 300 nucleotides, morepreferably at least 400 nucleotides, or most preferably at least 500nucleotides in length. Even longer probes may be used, e.g., nucleicacid probes which are at least 600 nucleotides, at least preferably atleast 700 nucleotides, more preferably at least 800 nucleotides, or mostpreferably at least 900 nucleotides in length. Both DNA and RNA probescan be used. The probes are typically labeled for detecting thecorresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).Such probes are encompassed by the present invention.

A genomic DNA or cDNA library prepared from such other organisms may,therefore, be screened for DNA which hybridizes with the probesdescribed above and which encodes a polypeptide having beta-glucosidaseactivity. Genomic or other DNA from such other organisms may beseparated by agarose or polyacrylamide gel electrophoresis, or otherseparation techniques. DNA from the libraries or the separated DNA maybe transferred to and immobilized on nitrocellulose or other suitablecarrier material. In order to identify a clone or DNA which ishomologous with SEQ ID NO: 1 or a subsequence thereof, the carriermaterial is preferably used in a Southern blot.

For purposes of the present invention, hybridization indicates that thenucleotide sequence hybridizes to a labeled nucleic acid probecorresponding to the mature polypeptide coding sequence of SEQ ID NO: 1,the cDNA sequence contained in the mature polypeptide coding sequence ofSEQ ID NO: 1; its complementary strand; or a subsequence thereof; underat least very low to very high stringency conditions. Molecules to whichthe nucleic acid probe hybridizes under these conditions can be detectedusing, for example, X-ray film.

In a preferred aspect, the nucleic acid probe is the mature polypeptidecoding sequence of SEQ ID NO: 1. In another preferred aspect, thenucleic acid probe is nucleotides 171 to 2753 of SEQ ID NO: 1. Inanother preferred aspect, the nucleic acid probe is a polynucleotidesequence which encodes the polypeptide of SEQ ID NO: 2, or a subsequencethereof. In another preferred aspect, the nucleic acid probe is SEQ IDNO: 1. In another preferred aspect, the nucleic acid probe is the maturepolypeptide coding sequence of SEQ ID NO: 1. In another preferredaspect, the nucleic acid probe is the mature polypeptide coding sequencecontained in plasmid pKKAB which is contained in E. coli NRRL B-30860.

For long probes of at least 100 nucleotides in length, very low to veryhigh stringency conditions are defined as prehybridization andhybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared anddenatured salmon sperm DNA, and either 25% formamide for very low andlow stringencies, 35% formamide for medium and medium-high stringencies,or 50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally.

For long probes of at least 100 nucleotides in length, the carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS preferably at least at 45° C. (very low stringency), morepreferably at least at 50° C. (low stringency), more preferably at leastat 55° C. (medium stringency), more preferably at least at 60° C.(medium-high stringency), even more preferably at least at 65° C. (highstringency), and most preferably at least at 70° C. (very highstringency).

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, stringency conditions are defined as prehybridization,hybridization, and washing post-hybridization at about 5° C. to about10° C. below the calculated T_(n), using the calculation according toBolton and McCarthy (1962, Proceedings of the National Academy ofSciences USA 48: 1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA,0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1 mMsodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per mlfollowing standard Southern blotting procedures for 12 to 24 hoursoptimally.

For short probes which are about 15 nucleotides to about 70 nucleotidesin length, the carrier material is washed once in 6×SCC plus 0.1% SDSfor 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10°C. below the calculated T_(m).

In a third aspect, the present invention relates to isolatedpolypeptides having beta-glucosidase activity comprisingA-E-[ST][IV][KR]G-[IM]-Q-[DS]-[ST]-G-V-[IV]-A.

In a fourth aspect, the present invention relates to artificial variantscomprising a conservative substitution, deletion, and/or insertion ofone or more amino acids of the mature polypeptide of SEQ ID NO: 2; or ahomologous sequence thereof. Preferably, amino acid changes are of aminor nature, that is conservative amino acid substitutions orinsertions that do not significantly affect the folding and/or activityof the protein; small deletions, typically of one to about 30 aminoacids; small amino- or carboxyl-terminal extensions, such as anamino-terminal methionine residue; a small linker peptide of up to about20-25 residues; or a small extension that facilitates purification bychanging net charge or another function, such as a poly-histidine tract,an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in the parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,beta-glucosidase activity) to identify amino acid residues that arecritical to the activity of the molecule. See also, Hilton et al., 1996,J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or otherbiological interaction can also be determined by physical analysis ofstructure, as determined by such techniques as nuclear magneticresonance, crystallography, electron diffraction, or photoaffinitylabeling, in conjunction with mutation of putative contact site aminoacids. See, for example, de Vos et al., 1992, Science 255: 306-312;Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992,FEBS Lett. 309: 59-64. The identities of essential amino acids can alsobe inferred from analysis of identities with polypeptides which arerelated to a polypeptide according to the invention.

Single or multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis, recombination, and/or shuffling, followedby a relevant screening procedure, such as those disclosed byReidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer,1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO95/22625. Other methods that can be used include error-prone PCR, phagedisplay (e.g., Lowman et al., 1991, Biochem. 30: 10832-10837; U.S. Pat.No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshireet al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide of interest, and can be applied to polypeptides of unknownstructure.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 2, such as aminoacids 37 to 878 of SEQ ID NO: 2, is 10, preferably 9, more preferably 8,more preferably 7, more preferably at most 6, more preferably 5, morepreferably 4, even more preferably 3, most preferably 2, and even mostpreferably 1.

Sources of Polypeptides Having Beta-Glucosidase Activity

A polypeptide having beta-glucosidase activity of the present inventionmay be obtained from microorganisms of any genus. For purposes of thepresent invention, the term “obtained from” as used herein in connectionwith a given source shall mean that the polypeptide encoded by anucleotide sequence is produced by the source or by a strain in whichthe nucleotide sequence from the source has been inserted. In apreferred aspect, the polypeptide obtained from a given source issecreted extracellularly.

A polypeptide of the present invention may be a bacterial polypeptide.For example, the polypeptide may be a gram positive bacterialpolypeptide such as a Bacillus, Streptococcus, Streptomyces,Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium,Geobacillus, or Oceanobacillus polypeptide having beta-glucosidaseactivity, or a Gram negative bacterial polypeptide such as an E. coli,Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium,Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma polypeptide havingbeta-glucosidase activity.

In a preferred aspect, the polypeptide is a Bacillus alkalophilus,Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans,Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillusthuringiensis polypeptide having beta-glucosidase activity.

In another preferred aspect, the polypeptide is a Streptococcusequisimilis, Streptococcus pyogenes, Streptococcus uberis, orStreptococcus equi subsp. Zooepidemicus polypeptide havingbeta-glucosidase activity.

In another preferred aspect, the polypeptide is a Streptomycesachromogenes, Streptomyces avermitilis, Streptomyces coelicolor,Streptomyces griseus, or Streptomyces lividans polypeptide havingbeta-glucosidase activity.

A polypeptide of the present invention may also be a fungal polypeptide,and more preferably a yeast polypeptide such as a Candida,Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowiapolypeptide having beta-glucosidase activity; or more preferably afilamentous fungal polypeptide such as an Acremonium, Aspergillus,Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocaffimastix, Neurospora,Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces,Thermoascus, Thielavia, Tolypocladium, or Trichoderma polypeptide havingbeta-glucosidase activity.

In a preferred aspect, the polypeptide is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis polypeptide havingbeta-glucosidase activity.

In another preferred aspect, the polypeptide is an Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, Aspergillus oryzae, Fusarium bactridioides, Fusarium cerealis,Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum,Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusariumoxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum,Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum,Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum,Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, Trichoderma viride, Thielavia achromatica, Thielaviaalbomyces, Thielavia albopilosa, Thielavia australeinsis, Thielaviafimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana,Thielavia spededonium, Thielavia setosa, Thielavia subthermophila,Thielavia terrestris, Thielavia terricola, Thielavia thermophila,Thielavia variospora, or Thielavia wareingii polypeptide havingbeta-glucosidase activity.

In another preferred aspect, the polypeptide is a Penicilliumbrasilianum, Penicillium camembertii, Penicillium capsulatum,Penicillium chrysogenum, Penicillium citreonigrum, Penicillium citrinum,Penicillium claviforme, Penicillium corylophilum, Penicillium crustosum,Penicillium digitatum, Penicillium expansum, Penicillium funiculosum,Penicillium glabrum, Penicillium granulatum, Penicillium griseofulvum,Penicillium islandicum, Penicillium italicum, Penicillium janthinellum,Penicillium lividum, Penicillium megasporum, Penicillium melinii,Penicillium notatum, Penicillium oxalicum, Penicillium puberulum,Penicillium purpurescens, Penicillium purpurogenum, Penicilliumroquefortii, Penicillium rugulosum, Penicillium spinulosum, Penicilliumwaksmanii, or Penicillium sp. polypeptide having beta-glucosidaseactivity.

In a more preferred aspect, the polypeptide is a Penicillium brasilianumpolypeptide having beta-glucosidase activity. In a most preferredaspect, the polypeptide is a Penicillium brasilianum IBT 20888polypeptide having beta-glucosidase activity, e.g., the polypeptide ofSEQ ID NO: 2 or the mature polypeptide thereof.

It will be understood that for the aforementioned species the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these species are readily accessible to the public in anumber of culture collections, such as the American Type CultureCollection (ATCC), Deutsche Sammlung von Mikroorganismen andZellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), andAgricultural Research Service Patent Culture Collection, NorthernRegional Research Center (NRRL).

Furthermore, such polypeptides may be identified and obtained from othersources including microorganisms isolated from nature (e.g., soil,composts, water, etc.) using the above-mentioned probes. Techniques forisolating microorganisms from natural habitats are well known in theart. The polynucleotide may then be obtained by similarly screening agenomic or cDNA library of such a microorganism. Once a polynucleotidesequence encoding a polypeptide has been detected with the probe(s), thepolynucleotide can be isolated or cloned by utilizing techniques whichare well known to those of ordinary skill in the art (see, e.g.,Sambrook et al., 1989, supra).

Polypeptides of the present invention also include fused polypeptides orcleavable fusion polypeptides in which another polypeptide is fused atthe N-terminus or the C-terminus of the polypeptide or fragment thereof.A fused polypeptide is produced by fusing a nucleotide sequence (or aportion thereof) encoding another polypeptide to a nucleotide sequence(or a portion thereof) of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator.

Examples of cleavage sites include, but are not limited to, a Kex2 sitewhich encodes the dipeptide Lys-Arg (Martin et al., 2003, J. Ind.Microbiol. Biotechnol. 3: 568-76; Svetina et al., 2000, J. Biotechnol.76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol.63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; andContreras et al., 1991, Biotechnology 9: 378-381), an Ile-(Glu orAsp)-Gly-Arg site, which is cleaved by a Factor Xa protease after thearginine residue (Eaton et al., 1986, Biochem. 25: 505-512); aAsp-Asp-Asp-Asp-Lys site, which is cleaved by an enterokinase after thelysine (Collins-Racie et al., 1995, Biotechnology 13: 982-987); aHis-Tyr-Glu site or His-Tyr-Asp site, which is cleaved by Genenase I(Carter et al., 1989, Proteins: Structure, Function, and Genetics 6:240-248); a Leu-Val-Pro-Arg-Gly-Ser site, which is cleaved by thrombinafter the Arg (Stevens, 2003, Drug Discovery World 4: 35-48); aGlu-Asn-Leu-Tyr-Phe-Gln-Gly site, which is cleaved by TEV protease afterthe Gln (Stevens, 2003, supra); and a Leu-Glu-Val-Leu-Phe-Gln-Gly-Prosite, which is cleaved by a genetically engineered form of humanrhinovirus 3C protease after the Gln (Stevens, 2003, supra).

Polynucleotides

The present invention also relates to isolated polynucleotidescomprising or consisting of a nucleotide sequence which encode apolypeptide having beta-glucosidase activity of the present invention.

In a preferred aspect, the nucleotide sequence comprises or consists ofSEQ ID NO: 1. In another preferred aspect, the nucleotide sequencecomprises or consists of the sequence contained in plasmid pKKAB whichis contained in E. coli NRRL B-30860. In another preferred aspect, thenucleotide sequence comprises or consists of the mature polypeptidecoding region of SEQ ID NO: 1. In another preferred aspect, thenucleotide sequence comprises or consists of nucleotides 171 to 2753 ofSEQ ID NO: 1. In another preferred aspect, the nucleotide sequencecomprises or consists of the mature polypeptide coding region containedin plasmid pKKAB which is contained in E. coli NRRL B-30860. The presentinvention also encompasses nucleotide sequences which encode apolypeptide comprising or consisting of the amino acid sequence of SEQID NO: 2 or the mature polypeptide thereof, which differ from SEQ ID NO:1 or the mature polypeptide coding sequence thereof by virtue of thedegeneracy of the genetic code. The present invention also relates tosubsequences of SEQ ID NO: 1 which encode fragments of SEQ ID NO: 2 thathave beta-glucosidase activity.

The present invention also relates to isolated polynucleotidescomprising or consisting of nucleotide sequences which encodepolypeptides having beta-glucosidase activity, wherein the polypeptidescomprise A-E-[ST][IV][KR]G-[IM]-Q-[DS]-[ST]-G-V-[IV]-A.

The present invention also relates to mutant polynucleotides comprisingat least one mutation in the mature polypeptide coding sequence of SEQID NO: 1, in which the mutant nucleotide sequence encodes the maturepolypeptide of SEQ ID NO: 2. In a preferred aspect, the maturepolypeptide is amino acids 37 to 878 of SEQ ID NO: 2.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides of the present invention from such genomic DNA can beeffected, e.g., by using the well known polymerase chain reaction (PCR)or antibody screening of expression libraries to detect cloned DNAfragments with shared structural features. See, e.g., Innis et al.,1990, PCR: A Guide to Methods and Application, Academic Press, New York.Other nucleic acid amplification procedures such as ligase chainreaction (LCR), ligated activated transcription (LAT) and nucleotidesequence-based amplification (NASBA) may be used. The polynucleotidesmay be cloned from a strain of Penicillium, or another or relatedorganism and thus, for example, may be an allelic or species variant ofthe polypeptide encoding region of the nucleotide sequence.

The present invention also relates to isolated polynucleotidescomprising or consisting of nucleotide sequences which have a degree ofidentity to the mature polypeptide coding sequence of SEQ ID NO: 1 of atleast 60%, preferably at least 65%, more preferably at least 70%, morepreferably at least 75%, more preferably at least 80%, more preferablyat least 85%, more preferably at least 90%, even more preferably atleast 95%, and most preferably at least 96%, 97%, 98%, or 99% identity,which encode an active polypeptide. In a preferred aspect, the maturepolypeptide coding sequence is nucleotides 171 to 2753 of SEQ ID NO: 1.

Modification of a nucleotide sequence encoding a polypeptide of thepresent invention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., artificialvariants that differ in specific activity, thermostability, pH optimum,or the like. The variant sequence may be constructed on the basis of thenucleotide sequence presented as the polypeptide encoding region of SEQID NO: 1, e.g., a subsequence thereof, and/or by introduction ofnucleotide substitutions which do not give rise to another amino acidsequence of the polypeptide encoded by the nucleotide sequence, butwhich correspond to the codon usage of the host organism intended forproduction of the enzyme, or by introduction of nucleotide substitutionswhich may give rise to a different amino acid sequence. For a generaldescription of nucleotide substitution, see, e.g., Ford et al., 1991,Protein Expression and Purification 2: 95-107.

It will be apparent to those skilled in the art that such substitutionscan be made outside the regions critical to the function of the moleculeand still result in an active polypeptide. Amino acid residues essentialto the activity of the polypeptide encoded by an isolated polynucleotideof the invention, and therefore preferably not subject to substitution,may be identified according to procedures known in the art, such assite-directed mutagenesis or alanine-scanning mutagenesis (see, e.g.,Cunningham and Wells, 1989, supra). In the latter technique, mutationsare introduced at every positively charged residue in the molecule, andthe resultant mutant molecules are tested for beta-glucosidase activityto identify amino acid residues that are critical to the activity of themolecule. Sites of substrate-enzyme interaction can also be determinedby analysis of the three-dimensional structure as determined by suchtechniques as nuclear magnetic resonance analysis, crystallography orphotoaffinity labeling (see, e.g., de Vos et al., 1992, supra; Smith etal., 1992, supra; Wlodaver et al., 1992, supra).

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention, which hybridize under at leastvery low stringency conditions, preferably at least low stringencyconditions, more preferably at least medium stringency conditions, morepreferably at least medium-high stringency conditions, even morepreferably at least high stringency conditions, and most preferably atleast very high stringency conditions with (i) the mature polypeptidecoding sequence of SEQ ID NO: 1, (ii) the cDNA sequence contained in themature polypeptide coding sequence of SEQ ID NO: 1, or (iii) acomplementary strand of (i) or (ii); or allelic variants andsubsequences thereof (Sambrook et al., 1989, supra), as defined herein.In a preferred aspect, the mature polypeptide coding sequence isnucleotides 171 to 2753 of SEQ ID NO: 1.

The present invention also relates to isolated polynucleotides obtainedby (a) hybridizing a population of DNA under at least very low, low,medium, medium-high, high, or very high stringency conditions with (i)the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the cDNAsequence contained in the mature polypeptide coding sequence of SEQ IDNO: 1, or (iii) a complementary strand of (i) or (ii); and (b) isolatingthe hybridizing polynucleotide, which encodes a polypeptide havingbeta-glucosidase activity. In a preferred aspect, the mature polypeptidecoding sequence is nucleotides 171 to 2753 of SEQ ID NO: 1.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisingan isolated polynucleotide of the present invention operably linked toone or more control sequences that direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences.

An isolated polynucleotide encoding a polypeptide of the presentinvention may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide'ssequence prior to its insertion into a vector may be desirable ornecessary depending on the expression vector. The techniques formodifying polynucleotide sequences utilizing recombinant DNA methods arewell known in the art.

The control sequence may be an appropriate promoter sequence, anucleotide sequence which is recognized by a host cell for expression ofa polynucleotide encoding a polypeptide of the present invention. Thepromoter sequence contains transcriptional control sequences whichmediate the expression of the polypeptide. The promoter may be anynucleotide sequence which shows transcriptional activity in the hostcell of choice including mutant, truncated, and hybrid promoters, andmay be obtained from genes encoding extracellular or intracellularpolypeptides either homologous or heterologous to the host cell.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention, especially in abacterial host cell, are the promoters obtained from the E. coli lacoperon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilislevansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM),Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacilluslicheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylBgenes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978,Proceedings of the National Academy of Sciences USA 75: 3727-3731), aswell as the tac promoter (DeBoer et al., 1983, Proceedings of theNational Academy of Sciences USA 80: 21-25). Further promoters aredescribed in “Useful proteins from recombinant bacteria” in ScientificAmerican, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus oryzaeTAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus nigerneutral alpha-amylase, Aspergillus niger acid stable alpha-amylase,Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucormiehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzaetriose phosphate isomerase, Aspergillus nidulans acetamidase, Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporumtrypsin-like protease (WO 96/00787), Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a hybrid of the promoters from the genes for Aspergillus nigerneutral alpha-amylase and Aspergillus oryzae triose phosphateisomerase); and mutant, truncated, and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionine (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, a sequence recognized by a host cell to terminatetranscription. The terminator sequence is operably linked to the 3′terminus of the nucleotide sequence encoding the polypeptide. Anyterminator which is functional in the host cell of choice may be used inthe present invention.

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus oryzae TAKA amylase, Aspergillus nigerglucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillusniger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C(CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be a suitable leader sequence, anontranslated region of an mRNA which is important for translation bythe host cell. The leader sequence is operably linked to the 5′ terminusof the nucleotide sequence encoding the polypeptide. Any leader sequencethat is functional in the host cell of choice may be used in the presentinvention.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′ terminus of the nucleotide sequence and which,when transcribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencewhich is functional in the host cell of choice may be used in thepresent invention.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatcodes for an amino acid sequence linked to the amino terminus of apolypeptide and directs the encoded polypeptide into the cell'ssecretory pathway. The 5′ end of the coding sequence of the nucleotidesequence may inherently contain a signal peptide coding region naturallylinked in translation reading frame with the segment of the codingregion which encodes the secreted polypeptide. Alternatively, the 5′ endof the coding sequence may contain a signal peptide coding region whichis foreign to the coding sequence. The foreign signal peptide codingregion may be required where the coding sequence does not naturallycontain a signal peptide coding region. Alternatively, the foreignsignal peptide coding region may simply replace the natural signalpeptide coding region in order to enhance secretion of the polypeptide.However, any signal peptide coding region which directs the expressedpolypeptide into the secretory pathway of a host cell of choice, i.e.,secreted into a culture medium, may be used in the present invention.

Effective signal peptide coding regions for bacterial host cells are thesignal peptide coding regions obtained from the genes for Bacillus NCIB11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase,Bacillus licheniformis subtilisin, Bacillus licheniformisbeta-lactamase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), and Bacillus subtilis prsA. Further signal peptides aredescribed by Simonen and Palva, 1993, Microbiological Reviews 57:109-137.

Effective signal peptide coding regions for filamentous fungal hostcells are the signal peptide coding regions obtained from the genes forAspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase,Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase,Humicola insolens cellulase, Humicola insolens endoglucanase V, andHumicola lanuginosa lipase.

In a preferred aspect, the signal peptide is amino acids 1 to 19 of SEQID NO: 2. In another preferred aspect, the signal peptide coding regionis nucleotides 6 to 62 of SEQ ID NO: 1 which encode amino acids 1 to 19of SEQ ID NO: 2.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding regions are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding region that codesfor an amino acid sequence positioned at the amino terminus of apolypeptide. The resultant polypeptide is known as a proenzyme orpropolypeptide (or a zymogen in some cases). A propolypeptide isgenerally inactive and can be converted to a mature active polypeptideby catalytic or autocatalytic cleavage of the propeptide from thepropolypeptide. The propeptide coding region may be obtained from thegenes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilisneutral protease (nprT), Saccharomyces cerevisiae alpha-factor,Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophilalaccase (WO 95/33836).

In a preferred aspect, the propeptide is amino acids 20 to 36 of SEQ IDNO: 2. In another preferred aspect, the propeptide coding region isnucleotides 63 to 170 of SEQ ID NO: 1, or the cDNA sequence thereof,which encode amino acids 20 to 36 of SEQ ID NO: 2.

Where both signal peptide and propeptide regions are present at theamino terminus of a polypeptide, the propeptide region is positionednext to the amino terminus of a polypeptide and the signal peptideregion is positioned next to the amino terminus of the propeptideregion.

It may also be desirable to add regulatory sequences which allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those which causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the TAKA alpha-amylase promoter,Aspergillus niger glucoamylase promoter, and Aspergillus oryzaeglucoamylase promoter may be used as regulatory sequences. Otherexamples of regulatory sequences are those which allow for geneamplification. In eukaryotic systems, these include the dihydrofolatereductase gene which is amplified in the presence of methotrexate, andthe metallothionein genes which are amplified with heavy metals. Inthese cases, the nucleotide sequence encoding the polypeptide would beoperably linked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleicacids and control sequences described herein may be joined together toproduce a recombinant expression vector which may include one or moreconvenient restriction sites to allow for insertion or substitution ofthe nucleotide sequence encoding the polypeptide at such sites.Alternatively, a nucleotide sequence of the present invention may beexpressed by inserting the nucleotide sequence or a nucleic acidconstruct comprising the sequence into an appropriate vector forexpression. In creating the expression vector, the coding sequence islocated in the vector so that the coding sequence is operably linkedwith the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the nucleotide sequence. The choice ofthe vector will typically depend on the compatibility of the vector withthe host cell into which the vector is to be introduced. The vectors maybe linear or closed circular plasmids.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids which togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon may be used.

The vectors of the present invention preferably contain one or moreselectable markers which permit easy selection of transformed,transfected, transduced, or the like cells. A selectable marker is agene the product of which provides for biocide or viral resistance,resistance to heavy metals, prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are the dal genes from Bacillussubtilis or Bacillus licheniformis, or markers which confer antibioticresistance such as ampicillin, kanamycin, chloramphenicol, ortetracycline resistance. Suitable markers for yeast host cells are ADE2,HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in afilamentous fungal host cell include, but are not limited to, amdS(acetamidase), argB (ornithine carbamoyltransferase), bar(phosphinothricin acetyltransferase), hph (hygromycinphosphotransferase), niaD (nitrate reductase), pyrG(orotidine-5′-phosphate decarboxylase), sC (sulfate adenyltransferase),and trpC (anthranilate synthase), as well as equivalents thereof.Preferred for use in an Aspergillus cell are the amdS and pyrG genes ofAspergillus nidulans or Aspergillus oryzae and the bar gene ofStreptomyces hygroscopicus.

The vectors of the present invention preferably contain an element(s)that permits integration of the vector into the host cell's genome orautonomous replication of the vector in the cell independent of thegenome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornonhomologous recombination. Alternatively, the vector may containadditional nucleotide sequences for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should preferably contain asufficient number of nucleic acids, such as 100 to 10,000 base pairs,preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000base pairs, which have a high degree of identity with the correspondingtarget sequence to enhance the probability of homologous recombination.The integrational elements may be any sequence that is homologous withthe target sequence in the genome of the host cell. Furthermore, theintegrational elements may be non-encoding or encoding nucleotidesequences. On the other hand, the vector may be integrated into thegenome of the host cell by non-homologous recombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication which functions in a cell.The term “origin of replication” or “plasmid replicator” is definedherein as a nucleotide sequence that enables a plasmid or vector toreplicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation ofthe AMA1 gene and construction of plasmids or vectors comprising thegene can be accomplished according to the methods disclosed in WO00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into the host cell to increase production of the gene product.An increase in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisingan isolated polynucleotide of the present invention, which areadvantageously used in the recombinant production of the polypeptides. Avector comprising a polynucleotide of the present invention isintroduced into a host cell so that the vector is maintained as achromosomal integrant or as a self-replicating extra-chromosomal vectoras described earlier. The term “host cell” encompasses any progeny of aparent cell that is not identical to the parent cell due to mutationsthat occur during replication. The choice of a host cell will to a largeextent depend upon the gene encoding the polypeptide and its source.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote.

Useful unicellular microorganisms are bacterial cells such as Grampositive bacteria and Gram negative bacteria. Gram positive bacteriainclude, but not limited to, Bacillus, Streptococcus, Streptomyces,Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium,Geobacillus, and Oceanobacillus. Gram negative bacteria include, but notlimited to, E. coli, Pseudomonas, Salmonella, Campylobacter,Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, andUreaplasma.

The bacterial host cell may be any Bacillus cell. Bacillus cells usefulin the practice of the present invention include, but are not limitedto, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis,Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillusfirmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis,Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus,Bacillus subtilis, and Bacillus thuringiensis cells.

In a preferred aspect, the bacterial host cell is a Bacillusamyloliquefaciens, Bacillus lentus, Bacillus licheniformis, Bacillusstearothermophilus or Bacillus subtilis cell. In a more preferredaspect, the bacterial host cell is a Bacillus amyloliquefaciens cell. Inanother more preferred aspect, the bacterial host cell is a Bacillusclausii cell. In another more preferred aspect, the bacterial host cellis a Bacillus licheniformis cell. In another more preferred aspect, thebacterial host cell is a Bacillus subtilis cell.

The bacterial host cell may be any Streptococcus cell. Streptococcuscells useful in the practice of the present invention include, but arenot limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus.

In another preferred aspect, the bacterial host cell is a Streptococcusequisimilis cell. In another preferred aspect, the bacterial host cellis a Streptococcus pyogenes cell. In another preferred aspect, thebacterial host cell is a Streptococcus uberis cell. In another preferredaspect, the bacterial host cell is a Streptococcus equi subsp.Zooepidemicus cell.

The bacterial host cell may be any Streptomyces cell. Streptomyces cellsuseful in the practice of the present invention include, but are notlimited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyceslividans.

In another preferred aspect, the bacterial host cell is a Streptomycesachromogenes cell. In another preferred aspect, the bacterial host cellis a Streptomyces avermitilis cell. In another preferred aspect, thebacterial host cell is a Streptomyces coelicolor cell. In anotherpreferred aspect, the bacterial host cell is a Streptomyces griseuscell. In another preferred aspect, the bacterial host cell is aStreptomyces lividans cell.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Molecular General Genetics 168: 111-115), by using competent cells (see,e.g., Young and Spizizin, 1961, Journal of Bacteriology 81: 823-829, orDubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56:209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988,Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler andThorne, 1987, Journal of Bacteriology 169: 5271-5278). The introductionof DNA into an E coli cell may, for instance, be effected by protoplasttransformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) orelectroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16:6127-6145). The introduction of DNA into a Streptomyces cell may, forinstance, be effected by protoplast transformation and electroporation(see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), byconjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171:3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc.Natl. Acad. Sci. USA 98:6289-6294). The introduction of DNA into aPseudomonas cell may, for instance, be effected by electroporation (see,e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or byconjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ.Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cellmay, for instance, be effected by natural competence (see, e.g., Perryand Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplasttransformation (see, e.g., Catt and Jollick, 1991, Microbios. 68:189-2070, by electroporation (see, e.g., Buckley et al., 1999, Appl.Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g.,Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method knownin the art can be used for introducing DNA into a host cell.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

In a preferred aspect, the host cell is a fungal cell. “Fungi” as usedherein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota,and Zygomycota (as defined by Hawksworth et al., In, Ainsworth andBisby's Dictionary of The Fungi, 8th edition, 1995, CAB International,University Press, Cambridge, UK) as well as the Oomycota (as cited inHawksworth et al., 1995, supra, page 171) and all mitosporic fungi(Hawksworth et al., 1995, supra).

In a more preferred aspect, the fungal host cell is a yeast cell.“Yeast” as used herein includes ascosporogenous yeast (Endomycetales),basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti(Blastomycetes). Since the classification of yeast may change in thefuture, for the purposes of this invention, yeast shall be defined asdescribed in Biology and Activities of Yeast (Skinner, F. A., Passmore,S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium SeriesNo. 9, 1980).

In an even more preferred aspect, the yeast host cell is a Candida,Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, orYarrowia cell.

In a most preferred aspect, the yeast host cell is a Saccharomycescarlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus,Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomycesnorbensis, or Saccharomyces oviformis cell. In another most preferredaspect, the yeast host cell is a Kluyveromyces lactis cell. In anothermost preferred aspect, the yeast host cell is a Yarrowia lipolyticacell.

In another more preferred aspect, the fungal host cell is a filamentousfungal cell. “Filamentous fungi” include all filamentous forms of thesubdivision Eumycota and Oomycota (as defined by Hawksworth et al.,1995, supra). The filamentous fungi are generally characterized by amycelial wall composed of chitin, cellulose, glucan, chitosan, mannan,and other complex polysaccharides. Vegetative growth is by hyphalelongation and carbon catabolism is obligately aerobic. In contrast,vegetative growth by yeasts such as Saccharomyces cerevisiae is bybudding of a unicellular thallus and carbon catabolism may befermentative.

In an even more preferred aspect, the filamentous fungal host cell is anAcremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis,Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola,Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora,Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

In a most preferred aspect, the filamentous fungal host cell is anAspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus,Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger orAspergillus oryzae cell. In another most preferred aspect, thefilamentous fungal host cell is a Fusarium bactridioides, Fusariumcerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsulphureum, Fusarium torulosum, Fusarium trichothecioides, or Fusariumvenenatum cell. In another most preferred aspect, the filamentous fungalhost cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsisaneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens,Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa,Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus,Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthorathermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaetechrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris,Trametes villosa, Trametes versicolor, Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238 023 and Yelton et al., 1984, Proceedings of the NationalAcademy of Sciences USA 81: 1470-1474. Suitable methods for transformingFusarium species are described by Malardier et al., 1989, Gene 78:147-156, and WO 96/00787. Yeast may be transformed using the proceduresdescribed by Becker and Guarente, In Abelson, J. N. and Simon, M. I.,editors, Guide to Yeast Genetics and Molecular Biology, Methods inEnzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Itoet al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978,Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

The present invention also relates to methods for producing apolypeptide of the present invention, comprising: (a) cultivating acell, which in its wild-type form is capable of producing thepolypeptide, under conditions conducive for production of thepolypeptide; and (b) recovering the polypeptide. Preferably, the cell isof the genus Penicillium, more preferably Penicillium brasilianum, andmost preferably Penicillium brasilianum IBT 20888.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising: (a) cultivating a hostcell under conditions conducive for production of the polypeptide; and(b) recovering the polypeptide.

The present invention also relates to methods for producing apolypeptide of the present invention, comprising: (a) cultivating a hostcell under conditions conducive for production of the polypeptide,wherein the host cell comprises a mutant nucleotide sequence having atleast one mutation in the mature polypeptide coding sequence of SEQ IDNO: 1, wherein the mutant nucleotide sequence encodes a polypeptidewhich consists of the mature polypeptide of SEQ ID NO: 2, and (b)recovering the polypeptide. In a preferred aspect, the maturepolypeptide is amino acids 37 to 878 of SEQ ID NO: 2.

In the production methods of the present invention, the cells arecultivated in a nutrient medium suitable for production of thepolypeptide using methods well known in the art. For example, the cellmay be cultivated by shake flask cultivation, and small-scale orlarge-scale fermentation (including continuous, batch, fed-batch, orsolid state fermentations) in laboratory or industrial fermentorsperformed in a suitable medium and under conditions allowing thepolypeptide to be expressed and/or isolated. The cultivation takes placein a suitable nutrient medium comprising carbon and nitrogen sources andinorganic salts, using procedures known in the art. Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). If the polypeptide is secreted into the nutrient medium,the polypeptide can be recovered directly from the medium. If thepolypeptide is not secreted into the medium, it can be recovered fromcell lysates.

The polypeptides may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide as described herein.

The resulting polypeptide may be recovered using methods known in theart. For example, the polypeptide may be recovered from the nutrientmedium by conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray-drying, evaporation, orprecipitation.

The polypeptides of the present invention may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobic,chromatofocusing, and size exclusion), electrophoretic procedures (e.g.,preparative isoelectric focusing), differential solubility (e.g.,ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g.,Protein Purification, J.-C. Janson and Lars Ryden, editors, VCHPublishers, New York, 1989) to obtain substantially pure polypeptides.

Plants

The present invention also relates to a transgenic plant, plant part, orplant cell which has been transformed with an isolated polynucleotideencoding a polypeptide having beta-glucosidase activity of the presentinvention so as to express and produce the polypeptide in recoverablequantities. The polypeptide may be recovered from the plant or plantpart. Alternatively, the plant or plant part containing the recombinantpolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilisation of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide of thepresent invention may be constructed in accordance with methods known inthe art. In short, the plant or plant cell is constructed byincorporating one or more expression constructs encoding a polypeptideof the present invention into the plant host genome or chloroplastgenome and propagating the resulting modified plant or plant cell into atransgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct whichcomprises a polynucleotide encoding a polypeptide of the presentinvention operably linked with appropriate regulatory sequences requiredfor expression of the nucleotide sequence in the plant or plant part ofchoice. Furthermore, the expression construct may comprise a selectablemarker useful for identifying host cells into which the expressionconstruct has been integrated and DNA sequences necessary forintroduction of the construct into the plant in question (the latterdepends on the DNA introduction method to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide of the present invention may be constitutive or inducible,or may be developmental, stage or tissue specific, and the gene productmay be targeted to a specific tissue or plant part such as seeds orleaves. Regulatory sequences are, for example, described by Tague etal., 1988, Plant Physiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294, Christensen et al., 1992, Plant Mo. Biol. 18: 675-689; Zhang etal., 1991, Plant Cell 3: 1155-1165). organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoterfrom the legumin B4 and the unknown seed protein gene from Vicia faba(Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), apromoter from a seed oil body protein (Chen et al., 1998, Plant and CellPhysiology 39: 935-941), the storage protein napA promoter from Brassicanapus, or any other seed specific promoter known in the art, e.g., asdescribed in WO 91/14772. Furthermore, the promoter may be a leafspecific promoter such as the rbcs promoter from rice or tomato (Kyozukaet al., 1993, Plant Physiology 102: 991-1000, the chlorella virusadenine methyltransferase gene promoter (Mitra and Higgins, 1994, PlantMolecular Biology 26: 85-93), or the aldP gene promoter from rice(Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or awound inducible promoter such as the potato pin2 promoter (Xu et al.,1993, Plant Molecular Biology 22: 573-588). Likewise, the promoter mayinducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide of the present invention in the plant. Forinstance, the promoter enhancer element may be an intron which is placedbetween the promoter and the nucleotide sequence encoding a polypeptideof the present invention. For instance, Xu et al., 1993, supra, disclosethe use of the first intron of the rice actin 1 gene to enhanceexpression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) andcan also be used for transforming monocots, although othertransformation methods are often used for these plants. Presently, themethod of choice for generating transgenic monocots is particlebombardment (microscopic gold or tungsten particles coated with thetransforming DNA) of embryonic calli or developing embryos (Christou,1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current OpinionBiotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10:667-674). An alternative method for transformation of monocots is basedon protoplast transformation as described by Omirulleh et al., 1993,Plant Molecular Biology 21: 415-428.

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well-known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs or sitespecific excision of the selection gene by a specific recombinase.

The present invention also relates to methods for producing apolypeptide of the present invention comprising: (a) cultivating atransgenic plant or a plant cell comprising a polynucleotide encoding apolypeptide having beta-glucosidase activity of the present inventionunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Removal or Reduction of Beta-Glucosidase Activity

The present invention also relates to methods for producing a mutant ofa parent cell, which comprises disrupting or deleting a polynucleotidesequence, or a portion thereof, encoding a polypeptide of the presentinvention, which results in the mutant cell producing less of thepolypeptide than the parent cell when cultivated under the sameconditions.

The mutant cell may be constructed by reducing or eliminating expressionof a nucleotide sequence encoding a polypeptide of the present inventionusing methods well known in the art, for example, insertions,disruptions, replacements, or deletions. In a preferred aspect, thenucleotide sequence is inactivated. The nucleotide sequence to bemodified or inactivated may be, for example, the coding region or a partthereof essential for activity, or a regulatory element required for theexpression of the coding region. An example of such a regulatory orcontrol sequence may be a promoter sequence or a functional partthereof, i.e., a part that is sufficient for affecting expression of thenucleotide sequence. Other control sequences for possible modificationinclude, but are not limited to, a leader, polyadenylation sequence,propeptide sequence, signal peptide sequence, transcription terminator,and transcriptional activator.

Modification or inactivation of the nucleotide sequence may be performedby subjecting the parent cell to mutagenesis and selecting for mutantcells in which expression of the nucleotide sequence has been reduced oreliminated. The mutagenesis, which may be specific or random, may beperformed, for example, by use of a suitable physical or chemicalmutagenizing agent, by use of a suitable oligonucleotide, or bysubjecting the DNA sequence to PCR generated mutagenesis. Furthermore,the mutagenesis may be performed by use of any combination of thesemutagenizing agents.

Examples of a physical or chemical mutagenizing agent suitable for thepresent purpose include ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine,nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formicacid, and nucleotide analogues.

When such agents are used, the mutagenesis is typically performed byincubating the parent cell to be mutagenized in the presence of themutagenizing agent of choice under suitable conditions, and screeningand/or selecting for mutant cells exhibiting reduced or no expression ofthe gene.

Modification or inactivation of the nucleotide sequence may beaccomplished by introduction, substitution, or removal of one or morenucleotides in the gene or a regulatory element required for thetranscription or translation thereof. For example, nucleotides may beinserted or removed so as to result in the introduction of a stop codon,the removal of the start codon, or a change in the open reading frame.Such modification or inactivation may be accomplished by site-directedmutagenesis or PCR generated mutagenesis in accordance with methodsknown in the art. Although, in principle, the modification may beperformed in vivo, i.e., directly on the cell expressing the nucleotidesequence to be modified, it is preferred that the modification beperformed in vitro as exemplified below.

An example of a convenient way to eliminate or reduce expression of anucleotide sequence by a cell is based on techniques of genereplacement, gene deletion, or gene disruption. For example, in the genedisruption method, a nucleic acid sequence corresponding to theendogenous nucleotide sequence is mutagenized in vitro to produce adefective nucleic acid sequence which is then transformed into theparent cell to produce a defective gene. By homologous recombination,the defective nucleic acid sequence replaces the endogenous nucleotidesequence. It may be desirable that the defective nucleotide sequencealso encodes a marker that may be used for selection of transformants inwhich the nucleotide sequence has been modified or destroyed. In aparticularly preferred aspect, the nucleotide sequence is disrupted witha selectable marker such as those described herein.

Alternatively, modification or inactivation of the nucleotide sequencemay be performed by established anti-sense or RNAi techniques using asequence complementary to the nucleotide sequence. More specifically,expression of the nucleotide sequence by a cell may be reduced oreliminated by introducing a sequence complementary to the nucleotidesequence of the gene that may be transcribed in the cell and is capableof hybridizing to the mRNA produced in the cell. Under conditionsallowing the complementary anti-sense nucleotide sequence to hybridizeto the mRNA, the amount of protein translated is thus reduced oreliminated.

The present invention further relates to a mutant cell of a parent cellwhich comprises a disruption or deletion of a nucleotide sequenceencoding the polypeptide or a control sequence thereof, which results inthe mutant cell producing less of the polypeptide or no polypeptidecompared to the parent cell.

The polypeptide-deficient mutant cells so created are particularlyuseful as host cells for the expression of homologous and/orheterologous polypeptides. Therefore, the present invention furtherrelates to methods for producing a homologous or heterologouspolypeptide comprising: (a) cultivating the mutant cell under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide. The term “heterologous polypeptides” is defined herein aspolypeptides which are not native to the host cell, a native protein inwhich modifications have been made to alter the native sequence, or anative protein whose expression is quantitatively altered as a result ofa manipulation of the host cell by recombinant DNA techniques.

In a further aspect, the present invention relates to a method forproducing a protein product essentially free of beta-glucosidaseactivity by fermentation of a cell which produces both a polypeptide ofthe present invention as well as the protein product of interest byadding an effective amount of an agent capable of inhibitingbeta-glucosidase activity to the fermentation broth before, during, orafter the fermentation has been completed, recovering the product ofinterest from the fermentation broth, and optionally subjecting therecovered product to further purification.

In a further aspect, the present invention relates to a method forproducing a protein product essentially free of beta-glucosidaseactivity by cultivating the cell under conditions permitting theexpression of the product, subjecting the resultant culture broth to acombined pH and temperature treatment so as to reduce thebeta-glucosidase activity substantially, and recovering the product fromthe culture broth. Alternatively, the combined pH and temperaturetreatment may be performed on an enzyme preparation recovered from theculture broth. The combined pH and temperature treatment may optionallybe used in combination with a treatment with an beta-glucosidaseinhibitor.

In accordance with this aspect of the invention, it is possible toremove at least 60%, preferably at least 75%, more preferably at least85%, still more preferably at least 95%, and most preferably at least99% of the beta-glucosidase activity. Complete removal ofbeta-glucosidase activity may be obtained by use of this method.

The combined pH and temperature treatment is preferably carried out at apH in the range of 9 to 10 and a temperature in the range of at least65° C. for a sufficient period of time to attain the desired effect,where typically, 10 to 30 minutes is sufficient.

The methods used for cultivation and purification of the product ofinterest may be performed by methods known in the art.

The methods of the present invention for producing an essentiallybeta-glucosidase-free product is of particular interest in theproduction of eukaryotic polypeptides, in particular fungal proteinssuch as enzymes. The enzyme may be selected from, e.g., an amylolyticenzyme, lipolytic enzyme, proteolytic enzyme, cellulytic enzyme,oxidoreductase, or plant cell-wall degrading enzyme. Examples of suchenzymes include an aminopeptidase, amylase, amyloglucosidase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,galactosidase, beta-galactosidase, glucoamylase, glucose oxidase,glucosidase, haloperoxidase, hemicellulase, invertase, isomerase,laccase, ligase, lipase, lyase, mannosidase, oxidase, pectinolyticenzyme, peroxidase, phytase, phenoloxidase, polyphenoloxidase,proteolytic enzyme, ribonuclease, transferase, transglutaminase, orxylanase. The beta-glucosidase-deficient cells may also be used toexpress heterologous proteins of pharmaceutical interest such ashormones, growth factors, receptors, and the like.

It will be understood that the term “eukaryotic polypeptides” includesnot only native polypeptides, but also those polypeptides, e.g.,enzymes, which have been modified by amino acid substitutions, deletionsor additions, or other such modifications to enhance activity,thermostability, pH tolerance and the like.

In a further aspect, the present invention relates to a protein productessentially free from beta-glucosidase activity which is produced by amethod of the present invention.

Compositions

The present invention also relates to compositions comprising anisolated polypeptide of the present invention. Preferably, thecompositions are enriched in such a polypeptide. The term “enriched”indicates that the beta-glucosidase activity of the composition has beenincreased, e.g., with an enrichment factor of at least 1.1.

The composition may comprise a polypeptide of the present invention asthe major enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase.

The additional enzyme(s) may be produced, for example, by amicroorganism belonging to the genus Aspergillus, preferably Aspergillusaculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillusfoetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, or Aspergillus oryzae; Fusarium, preferably Fusariumbactridioides, Fusarium cerealis, Fusarium crookwellense, Fusariumculmorum, Fusarium graminearum, Fusarium graminum, Fusariumheterosporum, Fusarium negundi, Fusarium oxysporum, Fusariumreticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum,Fusarium sulphureum, Fusarium toruloseum, Fusarium trichothecioides, orFusarium venenatum; Humicola, preferably Humicola insolens or Humicolalanuginosa; or Trichoderma, preferably Trichoderma harzianum,Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei,or Trichoderma viride.

The polypeptide compositions may be prepared in accordance with methodsknown in the art and may be in the form of a liquid or a drycomposition. For instance, the polypeptide composition may be in theform of a granulate or a microgranulate. The polypeptide to be includedin the composition may be stabilized in accordance with methods known inthe art.

Examples are given below of preferred uses of the polypeptidecompositions of the invention. The dosage of the polypeptide compositionof the invention and other conditions under which the composition isused may be determined on the basis of methods known in the art.

Uses

The present invention is also directed to methods for using thepolypeptides having beta-glucosidase activity, or compositions thereof,as described below.

Degradation of Biomass to Monosaccharides, Disaccharides, andPolysaccharides

The present invention also relates to methods for degrading orconverting a cellulosic material, comprising: treating the cellulosicmaterial with an effective amount of one or more cellulolytic proteinsin the presence of an effective amount of a polypeptide havingbeta-glucosidase activity.

The polypeptides and host cells of the present invention, as describedherein, may be used in the production of monosaccharides, disaccharides,and polysaccharides as chemical or fermentation feedstocks from biomassfor the production of ethanol, plastics, other products orintermediates. The polypeptides having beta-glucosidase activity may bein the form of a crude fermentation broth with or without the cellsremoved or in the form of a semi-purified or purified enzymepreparation. The beta-glucosidase protein may also be a monocomponentpreparation, a multicomponent protein preparation, or a combination ofmulticomponent and monocomponent protein preparations. Alternatively, ahost cell of the present invention may be used as a source of thepolypeptide having beta-glucosidase activity in a fermentation processwith the biomass. The host cell may also contain native or heterologousgenes that encode cellulolytic protein as well as other enzymes usefulin the processing of biomass. In particular, the polypeptides and hostcells of the present invention may be used to increase the value ofprocessing residues (dried distillers grain, spent grains from brewing,sugarcane bagasse, etc.) by partial or complete degradation of celluloseor hemicellulose.

Biomass can include, but is not limited to, wood resources, municipalsolid waste, wastepaper, crops, and crop residues (see, for example,Wiselogel et al., 1995, in Handbook on Bioethanol (Charles E. Wyman,editor), pp. 105-118, Taylor & Francis, Washington D.C.; Wyman, 1994,Bioresource Technology 50: 3-16; Lynd, 1990, Applied Biochemistry andBiotechnology 24/25: 695-719; Mosier et al., 1999, Recent Progress inBioconversion of Lignocellulosics, in Advances in BiochemicalEngineering/Biotechnology, T. Scheper, managing editor, Volume 65, pp.23-40, Springer-Verlag, New York).

The predominant polysaccharide in the primary cell wall of biomass iscellulose, the second most abundant is hemi-cellulose, and the third ispectin. The secondary cell wall, produced after the cell has stoppedgrowing, also contains polysaccharides and is strengthened by polymericlignin covalently cross-linked to hemicellulose. Cellulose is ahomopolymer of anhydrocellobiose and thus a linear beta-(1-4)-D-glucan,while hemicelluloses include a variety of compounds, such as xylans,xyloglucans, arabinoxylans, and mannans in complex branched structureswith a spectrum of substituents. Although generally polymorphous,cellulose is found in plant tissue primarily as an insoluble crystallinematrix of parallel glucan chains. Hemicelluloses usually hydrogen bondto cellulose, as well as to other hemicelluloses, which help stabilizethe cell wall matrix.

In the methods of the present invention, the cellulolytic protein may beany protein involved in the processing of cellulosic material toglucose, or hemicellulosic material to xylose, mannose, galactose, andarabinose, their polymers, or products derived from them as describedbelow. As mentioned above, a host cell of the present invention may beused as a source of the polypeptide having beta-glucosidase and as asource of native or heterologous cellulolytic protein as well as otherenzymes useful in the processing of biomass. The cellulolytic proteinmay also be a monocomponent preparation, e.g., a cellulase, amulticomponent preparation, e.g., endoglucanase, cellobiohydrolase, or acombination of multicomponent and monocomponent protein preparations.The cellulolytic proteins may have activity, i.e., hydrolyze cellulose,either in the acid, neutral, or alkaline pH-range.

The cellulolytic protein may be of fungal or bacterial origin, which maybe obtained or isolated and purified from microorganisms which are knownto be capable of producing cellulolytic enzymes, e.g., species ofBacillus, Pseudomonas, Humicola, Coprinus, Thielavia, Fusarium,Myceliophthora, Acremonium, Cephalosporium, Scytalidium, Penicillium orAspergillus (see, for example, EP 458162), especially those produced bya strain selected from the species Humicola insolens (reclassified asScytalidium thermophilum, see for example, U.S. Pat. No. 4,435,307),Coprinus cinereus, Fusarium oxysporum, Myceliophthora thermophila,Meripilus giganteus, Thielavia terrestris, Acremonium sp., Acremoniumpersicinum, Acremonium acremonium, Acremonium brachypenium, Acremoniumdichromosporum, Acremonium obclavatum, Acremonium pinkertoniae,Acremonium roseogriseum, Acremonium incoloratum, and Acremonium furatum;preferably from the species Humicola insolens DSM 1800, Fusariumoxysporum DSM 2672, Myceliophthora thermophila CBS 117.65,Cephalosporium sp. RYM-202, Acremonium sp. CBS 478.94, Acremonium sp.CBS 265.95, Acremonium persicinum CBS 169.65, Acremonium acremonium AHU9519, Cephalosporium sp. CBS 535.71, Acremonium brachypenium CBS 866.73,Acremonium dichromosporum CBS 683.73, Acremonium obclavatum CBS 311.74,Acremonium pinkertoniae CBS 157.70, Acremonium roseogriseum CBS 134.56,Acremonium incoloratum CBS 146.62, and Acremonium furatum CBS 299.70H.Cellulolytic proteins may also be obtained from Trichoderma(particularly Trichoderma viride, Trichoderma reesei, and Trichodermakoningii), alkalophilic Bacillus (see, for example, U.S. Pat. No.3,844,890 and EP 458162), and Streptomyces (see, for example, EP458162). Chemically modified or protein engineered mutants are included.

Especially suitable cellulolytic proteins are the alkaline or neutralcellulases. Examples of such cellulases are cellulases described in EP495,257, EP 531,372, WO 96/11262, WO 96/29397, WO 98/08940. Otherexamples are cellulase variants such as those described in WO 94/07998,EP 531,315, U.S. Pat. Nos. 4,435,307, 5,457,046, 5,648,263, 5,686,593,5,691,178, 5,763,254, 5,776,757, WO 89/09259, WO 95/24471, WO 98/12307,and PCT/DK98/00299.

The cellulolytic proteins used in the methods of the present inventionmay be produced by fermentation of the above-noted microbial strains ona nutrient medium containing suitable carbon and nitrogen sources andinorganic salts, using procedures known in the art (see, e.g., Bennett,J. W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, AcademicPress, Calif., 1991). Suitable media are available from commercialsuppliers or may be prepared according to published compositions (e.g.,in catalogues of the American Type Culture Collection). Temperatureranges and other conditions suitable for growth and cellulolytic proteinproduction are known in the art (see, e.g., Bailey, J. E., and Ollis, D.F., Biochemical Engineering Fundamentals, McGraw-Hill Book Company, NY,1986).

The fermentation can be any method of cultivation of a cell resulting inthe expression or isolation of a cellulolytic protein. Fermentation may,therefore, be understood as comprising shake flask cultivation andsmall- or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermenters performed in a suitable medium and under conditions allowingthe cellulolytic protein to be expressed or isolated.

The resulting cellulolytic proteins produced by the methods describedabove may be recovered from the fermentation medium by conventionalprocedures including, but not limited to, centrifugation, filtration,spray-drying, evaporation, or precipitation. The recovered protein maythen be further purified by a variety of chromatographic procedures,e.g., ion exchange chromatography, gel filtration chromatography,affinity chromatography, or the like.

Cellulolytic protein may hydrolyze or hydrolyzes carboxymethyl cellulose(CMC), thereby decreasing the viscosity of the incubation mixture. Theresulting reduction in viscosity may be determined by a vibrationviscosimeter (e.g., MIVI 3000 from Sofraser, France). Determination ofcellulase activity, measured in terms of Cellulase Viscosity Unit(CEVU), quantifies the amount of catalytic activity present in a sampleby measuring the ability of the sample to reduce the viscosity of asolution of carboxymethyl cellulose (CMC). The assay is performed at thetemperature and pH suitable for the cellulolytic protein and substrate.For Celluclast™ (Novozymes A/S, Bagsværd, Denmark) the assay is carriedout at 40° C. in 0.1 M phosphate pH 9.0 buffer for 30 minutes with CMCas substrate (33.3 g/L carboxymethyl cellulose Hercules 7 LFD) and anenzyme concentration of approximately 3.3-4.2 CEVU/ml. The CEVU activityis calculated relative to a declared enzyme standard, such as CELLUZYME™Standard 17-1194 (obtained from Novozymes A/S, Bagsværd, Denmark).

Examples of cellulolytic preparations suitable for use in the presentinvention include, for example, CELLUCLAST™ (available from NovozymesA/S) and NOVOZYM™ 188 (available from Novozymes A/S). Other commerciallyavailable preparations comprising cellulase which may be used includeCELLUZYME™, CEREFLO™ and ULTRAFLO™ (Novozymes A/S), LAMINEX™ andSPEZYME™ CP (Genencor Int.), and ROHAMENT™ 7069 W (Röhm GmbH). Thecellulase enzymes are added in amounts effective from about 0.001% toabout 5.0% wt. of solids, more preferably from about 0.025% to about4.0% wt. of solids, and most preferably from about 0.005% to about 2.0%wt. of solids.

As mentioned above, the cellulolytic proteins used in the methods of thepresent invention may be monocomponent preparations, i.e., a componentessentially free of other cellulolytic components. The single componentmay be a recombinant component, i.e., produced by cloning of a DNAsequence encoding the single component and subsequent cell transformedwith the DNA sequence and expressed in a host (see, for example, WO91/17243 and WO 91/17244). Other examples of monocomponent cellulolyticproteins include, but are not limited to, those disclosed inJP-07203960-A and WO-9206209. The host is preferably a heterologous host(enzyme is foreign to host), but the host may under certain conditionsalso be a homologous host (enzyme is native to host). Monocomponentcellulolytic proteins may also be prepared by purifying such a proteinfrom a fermentation broth.

Examples of monocomponent cellulolytic proteins useful in practicing themethods of the present invention include, but are not limited to,endoglucanase, cellobiohydrolase, and other enzymes useful in degradingcellulosic biomass.

The term “endoglucanase” is defined herein as an endo-1,4-beta-D-glucan4-glucanohydrolase (E.C. No. 3.2.1.4) which catalyses endohydrolysis of1,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (suchas carboxymethyl cellulose and hydroxyethyl cellulose), lichenin,beta-1,4 bonds in mixed beta-1,3 glucans such as cereal beta-D-glucansor xyloglucans, and other plant material containing cellulosiccomponents. For purposes of the present invention, endoglucanaseactivity is determined using carboxymethyl cellulose (CMC) hydrolysisaccording to the procedure of Ghose, 1987, Pure and Appl. Chem. 59:257-268. One unit of endoglucanase activity is defined as 1.0 mmole ofreducing sugars produced per minute at 50° C., pH 4.8.

The term “cellobiohydrolase” is defined herein as a 1,4-beta-D-glucancellobiohydrolase (E.C. 3.2.1.91), which catalyzes the hydrolysis of1,4-beta-D-glucosidic linkages in cellulose, cellooligosaccharides, orany beta-1,4-linked glucose containing polymer, releasing cellobiosefrom the reducing or non-reducing ends of the chain. For purposes of thepresent invention, cellobiohydrolase activity is determined according tothe procedures described by Lever et al., 1972, Anal. Biochem. 47:273-279 and by van Tilbeurgh et al., 1982, FEBS Letters 149: 152-156;van Tilbeurgh and Claeyssens, 1985, FEBS Letters 187: 283-288.

The polypeptides of the present invention are used in conjunction withcellulolytic proteins to degrade the cellulosic and/or hemicellulosiccomponents of the biomass substrate to sugars, as mentioned above (see,for example, Brigham et al., 1995, in Handbook on Bioethanol (Charles E.Wyman, editor), pp. 119-141, Taylor & Francis, Washington D.C.; Lee,1997, Journal of Biotechnology 56: 1-24). The methods of the presentinvention can further comprise recovering the degraded cellulosicmaterial, using methods conventional in the art.

The optimum amounts of a polypeptide having beta-glucosidase activityand of cellulolytic proteins depends on several factors including, butnot limited to, the mixture of component cellulolytic proteins, thecellulosic substrate, the concentration of cellulosic substrate, thepretreatment(s) of the cellulosic substrate, temperature, time, pH, andinclusion of fermenting organism (e.g., yeast for SimultaneousSaccharification and Fermentation). The term “cellulolytic proteins” isdefined herein as those proteins or mixtures of proteins shown as beingcapable of hydrolyzing or converting or degrading cellulose under theconditions tested. Their amounts are usually measured by a common assaysuch as BCA (bicinchoninic acid, P. K. Smith et al., 1985, Anal.Biochem. 150: 76), and the preferred amount added in proportion to theamount of biomass being hydrolyzed.

In a preferred aspect, the amount of polypeptide having beta-glucosidaseactivity per g of cellulosic material is about 0.01 to about 2.0 mg,preferably about 0.025 to about 1.5 mg, more preferably about 0.05 toabout 1.25 mg, more preferably about 0.075 to about 1.25 mg, morepreferably about 0.1 to about 1.25 mg, even more preferably about 0.15to about 1.25 mg, and most preferably about 0.25 to about 1.0 mg per gof cellulosic material.

In another preferred aspect, the amount of cellulolytic proteins per gof cellulosic material is about 0.5 to about 50 mg, preferably about 0.5to about 40 mg, more preferably about 0.5 to about 25 mg, morepreferably about 0.75 to about 20 mg, more preferably about 0.75 toabout 15 mg, even more preferably about 0.5 to about 10 mg, and mostpreferably about 2.5 to about 10 mg per g of cellulosic material.

The methods of the present invention may be used to process a cellulosicmaterial to many useful substances, e.g., organic products, chemicalsand fuels. In addition to ethanol, some commodity and specialtychemicals that can be produced from cellulose include xylose, acetone,acetate, glycine, lysine, organic acids (e.g., lactic acid),1,3-propanediol, butanediol, glycerol, ethylene glycol, furfural,polyhydroxyalkanoates, cis, cis-muconic acid, and animal feed (Lynd, L.R., Wyman, C. E., and Gerngross, T. U., 1999, Biocommodity Engineering,Biotechnol. Prog., 15: 777-793; Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212; and Ryu, D. D. Y., and Mandels, M., 1980, Cellulases:biosynthesis and applications, Enz. Microb. Technol., 2: 91-102).Potential coproduction benefits extend beyond the synthesis of multipleorganic products from fermentable carbohydrate. Lignin-rich residuesremaining after biological processing can be converted to lignin-derivedchemicals, or used for power production.

Conventional methods used to process the cellulosic material inaccordance with the methods of the present invention are well understoodto those skilled in the art. The methods of the present invention may beimplemented using any conventional biomass processing apparatusconfigured to operate in accordance with the invention.

Such an apparatus may include a batch-stirred reactor, a continuous flowstirred reactor with ultrafiltration, a continuous plug-flow columnreactor (Gusakov, A. V., and Sinitsyn, A. P., 1985, Kinetics of theenzymatic hydrolysis of cellulose: 1. A mathematical model for a batchreactor process, Enz. Microb. Technol. 7: 346-352), an attrition reactor(Ryu, S. K., and Lee, J. M., 1983, Bioconversion of waste cellulose byusing an attrition bioreactor, Biotechnol. Bioeng. 25: 53-65), or areactor with intensive stirring induced by an electromagnetic field(Gusakov, A. V., Sinitsyn, A. P., Davydkin, I. Y., Davydkin, V. Y.,Protas, O. V., 1996, Enhancement of enzymatic cellulose hydrolysis usinga novel type of bioreactor with intensive stirring induced byelectromagnetic field, Appl. Biochem. Biotechnol. 56: 141-153).

The conventional methods include, but are not limited to,saccharification, fermentation, separate hydrolysis and fermentation(SHF), simultaneous saccharification and fermentation (SSF),simultaneous saccharification and cofermentation (SSCF), hybridhydrolysis and fermentation (HHF), and direct microbial conversion(DMC).

SHF uses separate process steps to first enzymatically hydrolyzecellulose to glucose and then ferment glucose to ethanol. In SSF, theenzymatic hydrolysis of cellulose and the fermentation of glucose toethanol are combined in one step (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212). SSCF includes the cofermentation of multiple sugars (Sheehan,J., and Himmel, M., 1999, Enzymes, energy and the environment: Astrategic perspective on the U.S. Department of Energy's research anddevelopment activities for bioethanol, Biotechnol. Prog. 15: 817-827).HHF includes two separate steps carried out in the same reactor but atdifferent temperatures, i.e., high temperature enzymaticsaccharification followed by SSF at a lower temperature that thefermentation strain can tolerate. DMC combines all three processes(cellulase production, cellulose hydrolysis, and fermentation) in onestep (Lynd, L. R., Weimer, P. J., van Zyl, W. H., and Pretorius, I. S.,2002, Microbial cellulose utilization: Fundamentals and biotechnology,Microbiol. Mol. Biol. Reviews 66: 506-577).

“Fermentation” or “fermentation process” refers to any fermentationprocess or any process comprising a fermentation step. A fermentationprocess includes, without limitation, fermentation processes used toproduce fermentation products including alcohols (e.g., arabinitol,butanol, ethanol, glycerol, methanol, 1,3-propanediol, sorbitol, andxylitol); organic acids (e.g., acetic acid, acetonic acid, adipic acid,ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid,fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaricacid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid,malonic acid, oxalic acid, propionic acid, succinic acid, and xylonicacid); ketones (e.g., acetone); amino acids (e.g., aspartic acid,glutamic acid, glycine, lysine, serine, and threonine); gases (e.g.,methane, hydrogen (H₂), carbon dioxide (CO₂), and carbon monoxide (CO)).Fermentation processes also include fermentation processes used in theconsumable alcohol industry (e.g., beer and wine), dairy industry (e.g.,fermented dairy products), leather industry, and tobacco industry.

The present invention further relates to methods for producing asubstance, comprising: (a) saccharifying a cellulosic material with aneffective amount of one or more cellulolytic proteins in the presence ofan effective amount of a polypeptide having beta-glucosidase activity;(b) fermenting the saccharified cellulosic material of step (a) with oneor more fermenting microorganisms; and (c) recovering the substance fromthe fermentation. The polypeptide having beta-glucosidase activity maybe in the form of a crude fermentation broth with or without the cellsor in the form of a semi-purified or purified enzyme preparation. Thebeta-glucosidase protein may be a monocomponent preparation, amulticomponent protein preparation, or a combination of multicomponentand monocomponent protein preparations.

The substance can be any substance derived from the fermentation. In apreferred aspect, the substance is an alcohol. It will be understoodthat the term “alcohol” encompasses a substance that contains one ormore hydroxyl moieties. In a more preferred aspect, the alcohol isarabinitol. In another more preferred aspect, the alcohol is butanol. Inanother more preferred aspect, the alcohol is ethanol. In another morepreferred aspect, the alcohol is glycerol. In another more preferredaspect, the alcohol is methanol. In another more preferred aspect, thealcohol is 1,3-propanediol. In another more preferred aspect, thealcohol is sorbitol. In another more preferred aspect, the alcohol isxylitol. See, for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G.T., 1999, Ethanol production from renewable resources, in Advances inBiochemical Engineering/Biotechnology, Scheper, T., ed., Springer-VerlagBerlin Heidelberg, Germany, 65: 207-241; Silveira, M. M., and Jonas, R.,2002, The biotechnological production of sorbitol, Appl. Microbiol.Biotechnol. 59: 400-408; Nigam, P., and Singh, D., 1995, Processes forfermentative production of xylitol—a sugar substitute, ProcessBiochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi, N. and Blaschek, H.P., 2003, Production of acetone, butanol and ethanol by Clostridiumbeijerinckii BA101 and in situ recovery by gas stripping, World Journalof Microbiology and Biotechnology 19 (6): 595-603.

In another preferred aspect, the substance is an organic acid. Inanother more preferred aspect, the organic acid is acetic acid. Inanother more preferred aspect, the organic acid is acetonic acid. Inanother more preferred aspect, the organic acid is adipic acid. Inanother more preferred aspect, the organic acid is ascorbic acid. Inanother more preferred aspect, the organic acid is citric acid. Inanother more preferred aspect, the organic acid is 2,5-diketo-D-gluconicacid. In another more preferred aspect, the organic acid is formic acid.In another more preferred aspect, the organic acid is fumaric acid. Inanother more preferred aspect, the organic acid is glucaric acid. Inanother more preferred aspect, the organic acid is gluconic acid. Inanother more preferred aspect, the organic acid is glucuronic acid. Inanother more preferred aspect, the organic acid is glutaric acid. Inanother preferred aspect, the organic acid is 3-hydroxypropionic acid.In another more preferred aspect, the organic acid is itaconic acid. Inanother more preferred aspect, the organic acid is lactic acid. Inanother more preferred aspect, the organic acid is malic acid. Inanother more preferred aspect, the organic acid is malonic acid. Inanother more preferred aspect, the organic acid is oxalic acid. Inanother more preferred aspect, the organic acid is propionic acid. Inanother more preferred aspect, the organic acid is succinic acid. Inanother more preferred aspect, the organic acid is xylonic acid. See,for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediatedextractive fermentation for lactic acid production from cellulosicbiomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

In another preferred aspect, the substance is a ketone. It will beunderstood that the term “ketone” encompasses a substance that containsone or more ketone moieties. In another more preferred aspect, theketone is acetone. See, for example, Qureshi and Blaschek, 2003, supra.

In another preferred aspect, the substance is an amino acid. In anothermore preferred aspect, the organic acid is aspartic acid. In anothermore preferred aspect, the amino acid is glutamic acid. In another morepreferred aspect, the amino acid is glycine. In another more preferredaspect, the amino acid is lysine. In another more preferred aspect, theamino acid is serine. In another more preferred aspect, the amino acidis threonine. See, for example, Richard, A., and Margaritis, A., 2004,Empirical modeling of batch fermentation kinetics for poly(glutamicacid) production and other microbial biopolymers, Biotechnology andBioengineering 87 (4): 501-515.

In another preferred aspect, the substance is a gas. In another morepreferred aspect, the gas is methane. In another more preferred aspect,the gas is H₂. In another more preferred aspect, the gas is CO₂. Inanother more preferred aspect, the gas is CO. See, for example, Kataoka,N., A. Miya, and K. Kiriyama, 1997, Studies on hydrogen production bycontinuous culture system of hydrogen-producing anaerobic bacteria,Water Science and Technology 36 (6-7): 41-47; and Gunaseelan V. N. inBiomass and Bioenergy, Vol. 13 (1-2), pp. 83-114, 1997, Anaerobicdigestion of biomass for methane production: A review.

Production of a substance from cellulosic material typically requiresfour major steps. These four steps are pretreatment, enzymatichydrolysis, fermentation, and recovery. Exemplified below is a processfor producing ethanol, but it will be understood that similar processescan be used to produce other substances, for example, the substancesdescribed above.

Pretreatment. In the pretreatment or pre-hydrolysis step, the cellulosicmaterial is heated to break down the lignin and carbohydrate structure,solubilize most of the hemicellulose, and make the cellulose fractionaccessible to cellulolytic enzymes. The heating is performed eitherdirectly with steam or in slurry where a catalyst may also be added tothe material to speed up the reactions. Catalysts include strong acids,such as sulfuric acid and SO₂, or alkali, such as sodium hydroxide. Thepurpose of the pre-treatment stage is to facilitate the penetration ofthe enzymes and microorganisms. Cellulosic biomass may also be subjectto a hydrothermal steam explosion pre-treatment (See U.S. PatentApplication No. 20020164730).

Saccharification. In the enzymatic hydrolysis step, also known assaccharification, enzymes as described herein are added to thepretreated material to convert the cellulose fraction to glucose and/orother sugars. The saccharification is generally performed instirred-tank reactors or fermentors under controlled pH, temperature,and mixing conditions. A saccharification step may last up to 200 hours.Saccharification may be carried out at temperatures from about 30° C. toabout 65° C., in particular around 50° C., and at a pH in the rangebetween about 4 and about 5, especially around pH 4.5. To produceglucose that can be metabolized by yeast, the hydrolysis is typicallyperformed in the presence of a polypeptide having beta-glucosidaseactivity.

Fermentation. In the fermentation step, sugars, released from thecellulosic material as a result of the pretreatment and enzymatichydrolysis steps, are fermented to ethanol by a fermenting organism,such as yeast. The fermentation can also be carried out simultaneouslywith the enzymatic hydrolysis in the same vessel, again under controlledpH, temperature, and mixing conditions. When saccharification andfermentation are performed simultaneously in the same vessel, theprocess is generally termed simultaneous saccharification andfermentation or SSF.

Any suitable cellulosic substrate or raw material may be used in afermentation process of the present invention. The substrate isgenerally selected based on the desired fermentation product, i.e., thesubstance to be obtained from the fermentation, and the processemployed, as is well known in the art. Examples of substrates suitablefor use in the methods of present invention, includecellulose-containing materials, such as wood or plant residues or lowmolecular sugars DP1-3 obtained from processed cellulosic material thatcan be metabolized by the fermenting microorganism, and which may besupplied by direct addition to the fermentation medium.

The term “fermentation medium” will be understood to refer to a mediumbefore the fermenting microorganism(s) is (are) added, such as, a mediumresulting from a saccharification process, as well as a medium used in asimultaneous saccharification and fermentation process (SSF).

“Fermenting microorganism” refers to any microorganism suitable for usein a desired fermentation process. Suitable fermenting microorganismsaccording to the invention are able to ferment, i.e., convert, sugars,such as glucose, xylose, arabinose, mannose, galactose, oroligosaccharides directly or indirectly into the desired fermentationproduct. Examples of fermenting microorganisms include fungal organisms,such as yeast. Preferred yeast includes strains of the Saccharomycesspp., and in particular, Saccharomyces cerevisiae. Commerciallyavailable yeast include, e.g., Red Star®/™/Lesaffre Ethanol Red(available from Red Star/Lesaffre, USA) FALI (available fromFleischmann's Yeast, a division of Burns Philp Food Inc., USA),SUPERSTART (available from Alltech), GERT STRAND (available from GertStrand AB, Sweden) and FERMIOL (available from DSM Specialties).

In a preferred aspect, the yeast is a Saccharomyces spp. In a morepreferred aspect, the yeast is Saccharomyces cerevisiae. In another morepreferred aspect, the yeast is Saccharomyces distaticus. In another morepreferred aspect, the yeast is Saccharomyces uvarum. In anotherpreferred aspect, the yeast is a Kluyveromyces spp. In another morepreferred aspect, the yeast is Kluyveromyces marxianus. In another morepreferred aspect, the yeast is Kluyveromyces fragilis. In anotherpreferred aspect, the yeast is a Candida spp. In another more preferredaspect, the yeast is Candida pseudotropicalis. In another more preferredaspect, the yeast is Candida brassicae. In another preferred aspect, theyeast is a Clavispora spp. In another more preferred aspect, the yeastis Clavispora lusitaniae. In another more preferred aspect, the yeast isClavispora opuntiae. In another preferred aspect, the yeast is aPachysolen spp. In another more preferred aspect, the yeast isPachysolen tannophilus. In another preferred aspect, the yeast is aBretannomyces spp. In another more preferred aspect, the yeast isBretannomyces clausenii (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212).

Bacteria that can efficiently ferment glucose to ethanol include, forexample, Zymomonas mobilis and Clostridium thermocellum (Philippidis,1996, supra).

It is well known in the art that the organisms described above can alsobe used to produce other substances, as described herein.

The cloning of heterologous genes in Saccharomyces cerevisiae (Chen, Z.,Ho, N. W. Y., 1993, Cloning and improving the expression of Pichiastipitis xylose reductase gene in Saccharomyces cerevisiae, Appl.Biochem. Biotechnol. 39-40: 135-147; Ho, N. W. Y., Chen, Z, Brainard, A.P., 1998, Genetically engineered Saccharomyces yeast capable ofeffectively cofermenting glucose and xylose, Appl. Environ. Microbiol.64: 1852-1859), or in bacteria such as Escherichia coli (Beall, D. S.,Ohta, K., Ingram, L. O., 1991, Parametric studies of ethanol productionfrom xylose and other sugars by recombinant Escherichia coli, Biotech.Bioeng. 38: 296-303), Klebsiella oxytoca (Ingram, L. O., Gomes, P. F.,Lai, X., Moniruzzaman, M., Wood, B. E., Yomano, L. P., York, S. W.,1998, Metabolic engineering of bacteria for ethanol production,Biotechnol. Bioeng. 58: 204-214), and Zymomonas mobilis (Zhang, M.,Eddy, C., Deanda, K., Finkelstein, M., and Picataggio, S., 1995,Metabolic engineering of a pentose metabolism pathway in ethanologenicZymomonas mobilis, Science 267: 240-243; Deanda, K., Zhang, M., Eddy,C., and Picataggio, S., 1996, Development of an arabinose-fermentingZymomonas mobilis strain by metabolic pathway engineering, Appl.Environ. Microbiol. 62: 4465-4470) has led to the construction oforganisms capable of converting hexoses and pentoses to ethanol(cofermentation).

Yeast or another microorganism typically is added to the degradedcellulose or hydrolysate and the fermentation is ongoing for about 24 toabout 96 hours, such as about 35 to about 60 hours. The temperature istypically between about 26° C. to about 40° C., in particular at about32° C., and at about pH 3 to about pH 6, in particular around pH 4-5.

In a preferred aspect, yeast or another microorganism is applied to thedegraded cellulose or hydrolysate and the fermentation is ongoing forabout 24 to about 96 hours, such as typically 35-60 hours. In apreferred aspects, the temperature is generally between about 26 toabout 40° C., in particular about 32° C., and the pH is generally fromabout pH 3 to about pH 6, preferably around pH 4-5. Yeast or anothermicroorganism is preferably applied in amounts of approximately 10⁵ to10¹², preferably from approximately 10⁷ to 10¹⁰, especiallyapproximately 5×10⁷ viable count per ml of fermentation broth. During anethanol producing phase the yeast cell count should preferably be in therange from approximately 10⁷ to 10¹⁰, especially around approximately2×10⁸. Further guidance of using yeast for fermentation can be found in,e.g., “The Alcohol Textbook” (Editors K. Jacques, T. P. Lyons and D. R.Kelsall, Nottingham University Press, United Kingdom 1999), which ishereby incorporated by reference.

The most widely used process in the art is the simultaneoussaccharification and fermentation (SSF) process where there is noholding stage for the saccharification, meaning that yeast and enzymeare added together.

For ethanol production, following the fermentation the mash is distilledto extract the ethanol. The ethanol obtained according to the process ofthe invention may be used as, e.g., fuel ethanol; drinking ethanol,i.e., potable neutral spirits, or industrial ethanol.

A fermentation stimulator may be used in combination with any of theenzymatic processes described herein to further improve the fermentationprocess, and in particular, the performance of the fermentingmicroorganism, such as, rate enhancement and ethanol yield. A“fermentation stimulator” refers to stimulators for growth of thefermenting microorganisms, in particular, yeast. Preferred fermentationstimulators for growth include vitamins and minerals. Examples ofvitamins include multivitamins, biotin, pantothenate, nicotinic acid,meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid,riboflavin, and Vitamins A, B, C, D, and E. See, e.g., Alfenore et al.,Improving ethanol production and viability of Saccharomyces cerevisiaeby a vitamin feeding strategy during fed-batch process, Springer-Verlag(2002), which is hereby incorporated by reference. Examples of mineralsinclude minerals and mineral salts that can supply nutrients, e.g., P,K, Mg, S, Ca, Fe, Zn, Mn, and Cu.

Recovery. The alcohol is separated from the fermented cellulosicmaterial and purified by conventional methods of distillation. Ethanolwith a purity of up to about 96 vol. % ethanol can be obtained, whichcan be used as, for example, fuel ethanol, drinking ethanol, i.e.,potable neutral spirits, or industrial ethanol.

For other substances, any method known in the art can be used including,but not limited to, chromatography (e.g., ion exchange, affinity,hydrophobic, chromatofocusing, and size exclusion), electrophoreticprocedures (e.g., preparative isoelectric focusing), differentialsolubility (e.g., ammonium sulfate precipitation), SDS-PAGE,distillation, or extraction.

In the methods of the present invention, the cellulolytic protein(s) andbeta-glucosidase polypeptide(s) may be supplemented by one or moreadditional enzyme activities to improve the degradation of thecellulosic material. Preferred additional enzymes are hemicellulases,esterases (e.g., lipases, phospholipases, and/or cutinases), proteases,laccases, peroxidases, or mixtures thereof.

In the methods of the present invention, the additional enzyme(s) may beadded prior to or during fermentation, including during or after thepropagation of the fermenting microorganism(s).

The enzymes referenced herein may be derived or obtained from anysuitable origin, including, bacterial, fungal, yeast or mammalianorigin. The term “obtained” means herein that the enzyme may have beenisolated from an organism which naturally produces the enzyme as anative enzyme. The term “obtained” also means herein that the enzyme mayhave been produced recombinantly in a host organism, wherein therecombinantly produced enzyme is either native or foreign to the hostorganism or has a modified amino acid sequence, e.g., having one or moreamino acids which are deleted, inserted and/or substituted, i.e., arecombinantly produced enzyme which is a mutant and/or a fragment of anative amino acid sequence or an enzyme produced by nucleic acidshuffling processes known in the art. Encompassed within the meaning ofa native enzyme are natural variants and within the meaning of a foreignenzyme are variants obtained recombinantly, such as by site-directedmutagenesis or shuffling.

The enzymes may also be purified. The term “purified” as used hereincovers enzymes free from other components from the organism from whichit is derived. The term “purified” also covers enzymes free fromcomponents from the native organism from which it is obtained. Theenzymes may be purified, with only minor amounts of other proteins beingpresent. The expression “other proteins” relate in particular to otherenzymes. The term “purified” as used herein also refers to removal ofother components, particularly other proteins and most particularlyother enzymes present in the cell of origin of the enzyme of theinvention. The enzyme may be “substantially pure,” that is, free fromother components from the organism in which it is produced, that is, forexample, a host organism for recombinantly produced enzymes. In apreferred aspect, the enzymes are at least 75% (w/w), preferably atleast 80%, more preferably at least 85%, more preferably at least 90%,more preferably at least 95%, more preferably at least 96%, morepreferably at least 97%, even more preferably at least 98%, or mostpreferably at least 99% pure. In another preferred aspect, the enzyme is100% pure.

The enzymes used in the present invention may be in any form suitablefor use in the processes described herein, such as, for example, a crudefermentation broth with or without cells, a dry powder or granulate, anon-dusting granulate, a liquid, a stabilized liquid, or a protectedenzyme. Granulates may be produced, e.g., as disclosed in U.S. Pat. Nos.4,106,991 and 4,661,452, and may optionally be coated by process knownin the art. Liquid enzyme preparations may, for instance, be stabilizedby adding stabilizers such as a sugar, a sugar alcohol or anotherpolyol, and/or lactic acid or another organic acid according toestablished process. Protected enzymes may be prepared according to theprocess disclosed in EP 238,216.

Detergent Compositions

The isolated polypeptides having beta-glucosidase activity of thepresent invention may be added to and thus become a component of adetergent composition.

The detergent composition of the present invention may for example beformulated as a hand or machine laundry detergent composition includinga laundry additive composition suitable for pre-treatment of stainedfabrics and a rinse added fabric softener composition, or be formulatedas a detergent composition for use in general household hard surfacecleaning operations, or be formulated for hand or machine dishwashingoperations.

In a specific aspect, the present invention provides a detergentadditive comprising a polypeptide having beta-glucosidase of the presentinvention. The detergent additive as well as the detergent compositionmay comprise one or more other enzymes such as a protease, lipase,cutinase, an amylase, carbohydrase, cellulase, pectinase, mannanase,arabinase, galactanase, xylanase, oxidase, e.g., a laccase, and/orperoxidase.

In general the properties of the chosen enzyme(s) should be compatiblewith the selected detergent, (i.e., pH-optimum, compatibility with otherenzymatic and non-enzymatic ingredients, etc.), and the enzyme(s) shouldbe present in effective amounts.

Proteases: Suitable proteases include those of animal, vegetable ormicrobial origin. Microbial origin is preferred. Chemically modified orprotein engineered mutants are included. The protease may be a serineprotease or a metalloprotease, preferably an alkaline microbial proteaseor a trypsin-like protease. Examples of alkaline proteases aresubtilisins, especially those derived from Bacillus, e.g., subtilisinNovo, subtilisin Carlsberg, subtilisin 309, subtilisin 147 andsubtilisin 168 (described in WO 89/06279). Examples of trypsin-likeproteases are trypsin (e.g., of porcine or bovine origin) and theFusarium protease described in WO 89/06270 and WO 94/25583.

Examples of useful proteases are the variants described in WO 92/19729,WO 98/20115, WO 98/20116, and WO 98/34946, especially the variants withsubstitutions in one or more of the following positions: 27, 36, 57, 76,87, 97, 101, 104, 120, 123, 167, 170, 194, 206, 218, 222, 224, 235 and274.

Preferred commercially available protease enzymes include Alcalase™,Savinase™ Primase™, Duralase™, Esperase™, and Kannase™ (Novozymes A/S),Maxatase™, Maxacal™, Maxapem™, Properase™, Purafect™, Purafect OxP™,FN2™, and FN3™ (Genencor International Inc.).

Lipases: Suitable lipases include those of bacterial or fungal origin.Chemically modified or protein engineered mutants are included. Examplesof useful lipases include lipases from Humicola (synonym Thermomyces),e.g., from H. lanuginosa (T. lanuginosus) as described in EP 258 068 andEP 305 216 or from H. insolens as described in WO 96/13580, aPseudomonas lipase, e.g., from P. alcaligenes or P. pseudoalcaligenes(EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P.fluorescens, Pseudomonas sp. strain SD 705 (WO 95/06720 and WO96/27002), P. wisconsinensis (WO 96/12012), a Bacillus lipase, e.g.,from B. subtilis (Dartois et al., 1993, Biochemica et Biophysica Acta,1131, 253-360), B. stearothermophilus (JP 64/744992) or B. pumilus (WO91/16422).

Other examples are lipase variants such as those described in WO92/05249, WO 94/01541, EP 407 225, EP 260 105, WO 95/35381, WO 96/00292,WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079 and WO97/07202.

Preferred commercially available lipases include Lipolase™, Lipex™, andLipolase Ultra™ (Novozymes A/S).

Amylases: Suitable amylases (alpha and/or beta) include those ofbacterial or fungal origin. Chemically modified or protein engineeredmutants are included. Amylases include, for example, α-amylases obtainedfrom Bacillus, e.g., a special strain of Bacillus licheniformis,described in more detail in GB 1,296,839.

Examples of useful amylases are the variants described in WO 94/02597,WO 94/18314, WO 96/23873, and WO 97/43424, especially the variants withsubstitutions in one or more of the following positions: 15, 23, 105,106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243,264, 304, 305, 391, 408, and 444.

Commercially available amylases are Duramyl™, Termamyl™, Fungamyl™ andBAN™ (Novozymes A/S), Rapidase™ and Purastar™ (from GenencorInternational Inc.).

Cellulases: Suitable cellulases include those of bacterial or fungalorigin. Chemically modified or protein engineered mutants are included.Suitable cellulases include cellulases from the genera Bacillus,Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, or Trichodermae.g., the fungal cellulases produced from Humicola insolens,Myceliophthora thermophila and Fusarium oxysporum disclosed in U.S. Pat.Nos. 4,435,307, 5,648,263, 5,691,178, 5,776,757 and WO 89/09259.

Especially suitable cellulases are the alkaline or neutral cellulaseshaving color care benefits. Examples of such cellulases are cellulasesdescribed in EP 0 495 257, EP 0 531 372, WO 96/11262, WO 96/29397, WO98/08940. Other examples are cellulase variants such as those describedin WO 94/07998, EP 0 531 315, U.S. Pat. Nos. 5,457,046, 5,686,593,5,763,254, WO 95/24471, WO 98/12307 and PCT/DK98/00299.

Commercially available cellulases include Celluclast®, Celluzyme™, andCarezyme™ (Novozymes A/S), Clazinase™, and Puradax HA™ (GenencorInternational Inc.), and KAC-500(B)™ (Kao Corporation).

Peroxidases/Oxidases: Suitable peroxidases/oxidases include those ofplant, bacterial or fungal origin. Chemically modified or proteinengineered mutants are included. Examples of useful peroxidases includeperoxidases from Coprinus, e.g., from C. cinereus, and variants thereofas those described in WO 93/24618, WO 95/10602, and WO 98/15257.

Commercially available peroxidases include Guardzyme™ (Novozymes A/S).

The detergent enzyme(s) may be included in a detergent composition byadding separate additives containing one or more enzymes, or by adding acombined additive comprising all of these enzymes. A detergent additiveof the invention, i.e., a separate additive or a combined additive, canbe formulated, for example, as a granulate, liquid, slurry, etc.Preferred detergent additive formulations are granulates, in particularnon-dusting granulates, liquids, in particular stabilized liquids, orslurries.

Non-dusting granulates may be produced, e.g., as disclosed in U.S. Pat.Nos. 4,106,991 and 4,661,452 and may optionally be coated by methodsknown in the art. Examples of waxy coating materials are poly(ethyleneoxide) products (polyethyleneglycol, PEG) with mean molar weights of1000 to 20000; ethoxylated nonylphenols having from 16 to 50 ethyleneoxide units; ethoxylated fatty alcohols in which the alcohol containsfrom 12 to 20 carbon atoms and in which there are 15 to 80 ethyleneoxide units; fatty alcohols; fatty acids; and mono- and di- andtriglycerides of fatty acids. Examples of film-forming coating materialssuitable for application by fluid bed techniques are given in GB1483591. Liquid enzyme preparations may, for instance, be stabilized byadding a polyol such as propylene glycol, a sugar or sugar alcohol,lactic acid or boric acid according to established methods. Protectedenzymes may be prepared according to the method disclosed in EP 238,216.

The detergent composition of the invention may be in any convenientform, e.g., a bar, a tablet, a powder, a granule, a paste or a liquid. Aliquid detergent may be aqueous, typically containing up to 70% waterand 0-30% organic solvent, or non-aqueous.

The detergent composition comprises one or more surfactants, which maybe non-ionic including semi-polar and/or anionic and/or cationic and/orzwitterionic. The surfactants are typically present at a level of from0.1% to 60% by weight.

When included therein the detergent will usually contain from about 1%to about 40% of an anionic surfactant such as linearalkylbenzenesulfonate, alpha-olefinsulfonate, alkyl sulfate (fattyalcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate,alpha-sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid, orsoap.

When included therein the detergent will usually contain from about 0.2%to about 40% of a non-ionic surfactant such as alcohol ethoxylate,nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide,ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide,polyhydroxy alkyl fatty acid amide, or N-acyl N-alkyl derivatives ofglucosamine (“glucamides”).

The detergent may contain 0-65% of a detergent builder or complexingagent such as zeolite, diphosphate, triphosphate, phosphonate,carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraaceticacid, diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinicacid, soluble silicates, or layered silicates (e.g., SKS-6 fromHoechst).

The detergent may comprise one or more polymers. Examples arecarboxymethylcellulose, poly(vinylpyrrolidone), poly(ethylene glycol),poly(vinyl alcohol), poly(vinylpyridine-N-oxide), poly(vinylimidazole),polycarboxylates such as polyacrylates, maleic/acrylic acid copolymers,and lauryl methacrylate/acrylic acid copolymers.

The detergent may contain a bleaching system which may comprise a H₂O₂source such as perborate or percarbonate which may be combined with aperacid-forming bleach activator such as tetraacetylethylenediamine ornonanoyloxybenzenesulfonate. Alternatively, the bleaching system maycomprise peroxyacids of, for example, the amide, imide, or sulfone type.

The enzyme(s) of the detergent composition of the invention may bestabilized using conventional stabilizing agents, e.g., a polyol such aspropylene glycol or glycerol, a sugar or sugar alcohol, lactic acid,boric acid, or a boric acid derivative, e.g., an aromatic borate ester,or a phenyl boronic acid derivative such as 4-formylphenyl boronic acid,and the composition may be formulated as described in, for example, WO92/19709 and WO 92/19708.

The detergent may also contain other conventional detergent ingredientssuch as, e.g., fabric conditioners including clays, foam boosters, sudssuppressors, anti-corrosion agents, soil-suspending agents, anti-soilredeposition agents, dyes, bactericides, optical brighteners,hydrotropes, tarnish inhibitors, or perfumes.

In the detergent compositions any enzyme, in particular the enzyme ofthe invention, may be added in an amount corresponding to 0.01-100 mg ofenzyme protein per liter of wash liquor, preferably 0.05-5 mg of enzymeprotein per liter of wash liquor, in particular 0.1-1 mg of enzymeprotein per liter of wash liquor.

The enzyme of the invention may additionally be incorporated in thedetergent formulations disclosed in WO 97/07202, which is herebyincorporated as reference.

Other Uses

The polypeptides having beta-glucosidase activity of the presentinvention may also be used in combination with other glycohydrolases andrelated enzymes, as described herein, in the treatment of textiles asbiopolishing agents and for reducing of fuzz, pilling, texturemodification, and stonewashing (N. K. Lange, in P. Suominen, T.Reinikainen (Eds.), Trichoderma reesei Cellulases and Other Hydrolases,Foundation for Biotechnical and Industrial Fermentation Research,Helsinki, 1993, pp. 263-272). In addition, the described polypeptidesmay also be used in combination with other glycohydrolases and relatedenzymes, as described herein, in wood processing for biopulping ordebarking, paper manufacturing for fiber modification, bleaching, andreduction of refining energy costs, whitewater treatment, important towastewater recycling, lignocellulosic fiber recycling such as deinkingand secondary fiber processing, and wood residue utilization (S. D,Mansfield and A. R. Esteghlalian in S. D, Mansfield and J. N. Saddler(Eds.), Applications of Enzymes to Lignocellulosics, ACS SymposiumSeries 855, Washington, D.C., 2003, pp. 2-29).

Signal Peptide and Propeptide

The present invention also relates to isolated polynucleotides encodinga signal peptide comprising or consisting of amino acids 1 to 19 of SEQID NO: 2. The present invention also relates to isolated polynucleotidesencoding a propeptide comprising or consisting of amino acids 20 to 36of SEQ ID NO: 2. The present invention also relates to isolatedpolynucleotides encoding a prepropeptide comprising or consisting ofamino acids 1 to 36 of SEQ ID NO: 2. In a preferred aspect, the signalpeptide is encoded by a polynucleotide that comprises or consists ofnucleotides 6 to 62 of SEQ ID NO: 1. In another preferred aspect, thepropeptide is encoded by a polynucleotide that comprises or consists ofnucleotides 63 to 170 of SEQ ID NO: 1 or the cDNA sequence thereof. Inanother preferred aspect, the prepropeptide is encoded by apolynucleotide that comprises or consists of nucleotides 1 to 108 of SEQID NO: 1.

The present invention also relates to nucleic acid constructs comprisinga gene encoding a protein operably linked to one or both of a firstnucleotide sequence encoding a signal peptide comprising or consistingof amino acids 1 to 19 of SEQ ID NO: 2, which allows secretion of theprotein into a culture medium, and a second nucleotide sequence encodinga propeptide comprising or consisting of amino acids 20 to 36 of SEQ IDNO: 2, wherein the gene is foreign to the first and second nucleotidesequences.

In a preferred aspect, the first nucleotide sequence comprises orconsists of nucleotides 6 to 62 of SEQ ID NO: 1. In another preferredaspect, the second nucleotide sequence comprises or consists ofnucleotides 63 to 170 of SEQ ID NO: 1 or the cDNA sequence thereof.

The present invention also relates to recombinant expression vectors andrecombinant host cells comprising such nucleic acid constructs.

The present invention also relates to methods for producing a proteincomprising: (a) cultivating such a recombinant host cell underconditions suitable for production of the protein; and (b) recoveringthe protein.

The first and second nucleotide sequences may be operably linked toforeign genes individually with other control sequences or incombination with other control sequences. Such other control sequencesare described supra. As described earlier, where both signal peptide andpropeptide regions are present at the amino terminus of a protein, thepropeptide region is positioned next to the amino terminus of a proteinand the signal peptide region is positioned next to the amino terminusof the propeptide region.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “protein” also encompasses two or more polypeptidescombined to form the encoded product. The proteins also include hybridpolypeptides which comprise a combination of partial or completepolypeptide sequences obtained from at least two different proteinswherein one or more may be heterologous or native to the host cell.Proteins further include naturally occurring allelic and engineeredvariations of the above mentioned proteins and hybrid proteins.

Preferably, the protein is a hormone or variant thereof, enzyme,receptor or portion thereof, antibody or portion thereof, or reporter.In a more preferred aspect, the protein is an oxidoreductase,transferase, hydrolase, lyase, isomerase, or ligase. In an even morepreferred aspect, the protein is an aminopeptidase, amylase,carbohydrase, carboxypeptidase, catalase, cellulase, chitinase,cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase,mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following exampleswhich should not be construed as limiting the scope of the invention.

EXAMPLES

Materials

Chemicals used as buffers and substrates were commercial products of atleast reagent grade.

DNA Sequencing

DNA sequencing was performed using an Applied Biosystems Model 3130×Genetic Analyzer (Applied Biosystems, Foster City, Calif., USA) usingdye terminator chemistry (Giesecke et al., 1992, Journal of Virol.Methods 38: 47-60). Sequences were assembled using phred/phrap/consed(University of Washington, Seattle, Wash., USA) with sequence specificprimers.

Strains

Penicillium brasilianum strain IBT 20888 (IBT Culture Collection ofFungi, Technical University of Denmark, Copenhagen, Denmark) was used asthe source of beta-glucosidase. Aspergillus oryzae BECH2 (WO 00/30322)was used for expression of the Penicillium brasilianum strain IBT 20888beta-glucosidase.

Media and Solutions

TE was composed of 10 mM Tris-1 mM EDTA.

LB medium was composed per liter of 10 g of tryptone, 5 g of yeastextract, and 5 g of sodium chloride.

LB plates were composed per liter of 10 g of tryptone, 5 g of yeastextract, 5 g of sodium chloride, and 15 g of Bacto Agar.

STC was composed of 1.2 M sorbitol, 10 mM Tris-HCl, and 10 mM CaCl₂, pH7.5.

PEG solution was composed of 60% PEG 4000, 10 mM Tris-HCl, and 10 mMCaCl₂, pH 7.5.

YPM medium was composed per liter of 10 g of yeast extract, 20 g ofpeptone, and 2% maltose.

Example 1 Isolation of Genomic DNA from Penicillium brasilianum

Spores of Penicillium brasilianum strain IBT 20888 were propagated onrice according to Carlsen, 1994, Ph.D. thesis, Department ofBiotechnology, The Technical University of Denmark. The spores wererecovered with 20 ml of 0.1% Tween 20 and inoculated at a concentrationof 1×10⁶ spores per ml into 100 ml of Mandels and Weber medium (Mandelsand Weber, 1969, Adv. Chem. Ser. 95: 394-414) containing 1% glucosesupplemented per liter with 0.25 g of yeast extract and 0.75 g ofBactopeptone in a 500 ml baffled shake flask. The fungal mycelia wereharvested after 24 hours of aerobic growth at 30° C., 150 rpm.

Mycelia were collected by filtration through a Nalgene DS0281-5000filter (Nalge Nunc International Corporation, Rochester, N.Y., USA)until dryness and frozen in liquid nitrogen. The frozen mycelia wereground to a powder in a dry ice chilled mortar and distributed to ascrew-cap tube. The powder was suspended in a total volume of 40 ml of50 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS)-NaOH pH 11buffer containing 0.5% lithium dodecyl sulfate and 0.5 mM EDTA. Thesuspension was placed at 60° C. for 2 hours and periodically resuspendedby inversion. To the suspension was added an equal volume ofphenol:chloroform (1:1 v/v) neutralized with 0.1 M Tris base, and thetube was mixed on a rotating wheel at 37° C. for 2 hours. Aftercentrifugation at 2500 rpm for 10 minutes in a Sorvall H1000B rotor, theaqueous phase (top phase) was re-extracted again with phenol:chloroform(1:1 v/v) and centrifuged at 15,000×g for 5 minutes. The aqueous phasefrom the second extraction was brought to 2.5 M ammonium acetate (stock10 M) and placed at −20° C. until frozen. After thawing, the extract wascentrifuged at 15,000×g for 20 minutes in a cold rotor. The pellet(primarily rRNA) was discarded and the nucleic acids in the supernatantwere precipitated by addition of 0.7 volumes of isopropanol. Aftercentrifugation at 15,000×g for 15 minutes, the pellet was rinsed threetimes with 5 ml of 70% ethanol (without resuspension), air-dried almostcompletely, and dissolved in 1.0 ml of 0.1×TE. The dissolved pellet wastransferred to two 1.5 ml microfuge tubes. The pellet solution wasprecipitated by addition of ammonium acetate (0.125 ml) to 2.0 M andethanol to 63% (1.07 ml) and centrifuged at maximum speed for 10 minutesin a Sorvall MC 12V microcentrifuge (Kendro Laboratory Products,Asheville, N.C., USA). The pellet was rinsed twice with 70% ethanol,air-dried completely, and dissolved in 500 μl of 0.1×TE.

Example 2 Preparation of a Genomic DNA Library

Genomic libraries were constructed using a TOPO Shotgun Subcloning Kit(Invitrogen, Carlsbad, Calif., USA). Briefly, total cellular DNA wassheared by nebulization under 10 psi nitrogen for 15 seconds andsize-fractionated on 1% agarose gels using 40 mM Tris base-20 mM sodiumacetate-1 mM disodium EDTA (TAE) buffer. DNA fragments migrating in thesize range 3-6 kb were excised and eluted using a MiniElute™ GelExtraction Kit (QIAGEN Inc, Valencia, Calif., USA). The eluted fragmentswere size-fractionated again using a 1% agarose gel as above and DNAfragments migrating in the size range 3-6 kb were excised and elutedusing a MiniElute™ Gel Extraction Kit.

The eluted DNA fragments were blunt end repaired and dephosphorylatedusing shrimp alkaline phosphatase (Roche Applied Science, Manheim,Germany). The blunt end DNA fragments were cloned into a pCR4Blunt-TOPOvector (Invitrogen, Carlsbad, Calif., USA) according to themanufacturer's instructions, transformed into electrocompetent E. coliTOP10 cells by electroporation, and plated on LB plates supplementedwith 100 μg of ampicillin per ml. The electroporation resulted in 15,300clones.

Example 3 Purification of Penicillium brasilianum Beta-glucosidase

Penicillium brasilianum strain IBT 20888 was grown in 4 liters ofMandels and Weber medium (Mandels and Weber, 1969, supra) in a 5 literbioreactor supplemented per liter with 1 g of yeast extract, 3 g ofbactopeptone, 30 g of cellulose, and 10 g of xylan. Spores werepropagated on rice according to Carlsen, 1994, supra. The bioreactor wasinoculated at a concentration of 1×10⁶ spores per ml. The pH wasmaintained at 5.0 by addition of either 2 M NH₄OH or 2 M HCl. Thetemperature was maintained at 30° C. The aeration was 4 liters perminute and 300-500 rpm. After 111 hours, the cultivation was terminatedand the broth was filtered through a glass fiber filter (GD 120,Advantec, Japan).

Beta-glucosidase activity was measured at room temperature in 50 mMsodium citrate pH 4.8. The substrate was 1 mM4-nitrophenyl-beta-D-glucopyranoside in 50 mM sodium citrate pH 4.8. Thebeta-glucosidase hydrolyzes the agluconic bond between 4-nitrophenol andglucose. The liberated 4-nitrophenol is yellow in alkaline solution andcan be determined spectrophotometrically at 405 nm. One internationalunit of activity (U) is defined as the amount of enzyme liberating 1μmole of 4-nitrophenol per minute at pH 4.8, 25° C.

Protein concentration was determined by SDS-PAGE. Fifteen μl of samplewas added to 15 μl of SDS-PAGE sample buffer (1.17 M sucrose, 1 MTris-HCL, pH 8.5, 278 mM SDS, 2.05 mM EDTA, 0.88 mM Brilliant Blue G,and 0.2 M dithiothreitol) in an Eppendorf tube and heated to 70° C. for10 minutes. Following heating the diluted sample was applied to aprecast 4-12% Bis-Tris pre-cast gel (Invitrogen, Groningen, TheNetherlands). In addition, a Mark 12 protein standard mixture(Invitrogen, Carlsbad, Calif., USA) was applied to the gel.

The gel was run in an Xcell SureLock™ gel apparatus (Invitrogen,Carlsbad, Calif., USA) for 50 minutes at 200 V. The running buffer wasmade by a 20-fold dilution of the standard buffer (1 M MOPS, 1 M TRIS,and 1% SDS). A 0.5 ml volume of NuPAGE® Antioxidant (Invitrogen,Carlsbad, Calif., USA) was added to the upper (cathode) buffer chamber.Following electrophoresis the gel was incubated for 60 minutes in astaining solution consisting of 0.1% (w/v) Coomassie Brilliant BlueR-250 dissolved in 10% acetic acid, 40% methanol, and 50% H₂O.Destaining of the gel was performed in 10% acetic acid, 30% methanol,and 60% H₂O.

Before purification, the filtrate was concentrated and buffer exchangedto 20 mM triethanolamine (TEA)-HCl pH 7.5 using an Amiconultrafiltration unit equipped with a PM10 membrane with 10 kDa cut-off(Millipore, Bedford, Mass., USA). The enzyme purification was performedat room temperature using a FPLC system (Amersham Bioscience, Uppsala,Sweden). Between each purification step, the buffer was exchanged in thepooled fractions to the sample buffer using either an Amiconultrafiltration unit or a 3.5 ml Microsep ultrafiltration unit with a 10kDa cut-off (Pall Life Sciences, Ann Arbor, Mich., USA). Elution of thebeta-glucosidase was monitored at 280 nm.

The retentate (38.5 ml) was loaded onto a XK 26 column packed with 75 mlof Q Sepharose HP (Amersham Bioscience, Uppsala, Sweden). The column waswashed with 180 ml of sample buffer. The sample buffer was 20 mM TEA-HClpH 7.5. The enzyme was eluted with a gradient up to 50% (over 800 ml) of20 mM TEA-HCl pH 7.5 with 1 M NaCl. Fractions of 10 ml were collected,assayed for beta-glucosidase activity, and fractions 81 to 85 werepooled.

The retentate (2.0 ml) from the previous step was loaded onto a Superdex75 10/300 GL column (Amersham Bioscience, Uppsala, Sweden) using 100 mMNaCH₃CO₂ pH 4.8 with 200 mM NaCl as the sample buffer. The enzyme waseluted with 60 ml of the same buffer. Fractions of 2 ml were collected,assayed for beta-glucosidase activity, and fractions 6 to 9 were pooledbased on activity and purity (SDS-PAGE).

The retentate (14 ml) from the Superdex 75 step was loaded onto a 6 mlRESOURCE Q column (Amersham Bioscience, Uppsala, Sweden) using 10 mMNaCH₃CO₂ pH 4.8 as the sample buffer. The column was washed with 30 mlof sample buffer. The enzyme was eluted with a gradient up to 50% (over180 ml) of 500 mM NaCH₃CO₂ pH 4.8. Fractions of 2 ml were collected,assayed for beta-glucosidase activity, and fractions 49 to 61 werepooled based on activity and purity (SDS-PAGE).

The retentate (12 ml) from the RESOURCE Q step was loaded onto another 6ml RESOURCE Q column (Amersham Bioscience, Uppsala, Sweden) using 10 mMNaCH₃CO₂ pH 4.8 as the sample buffer. The column was washed with 30 mlof sample buffer. The enzyme was eluted with a gradient up to 50% (over300 ml) of 500 mM NaCH₃CO₂ pH 4.8. Fractions of 2 ml were collected,assayed for beta-glucosidase activity, and fractions 63 to 67 werepooled based on specific activity and purity (SDS-PAGE).

The retentate (10.5 ml) from the RESOURCE Q step was loaded onto a 10 mlSource S column (Amersham Bioscience, Uppsala, Sweden) using 10 mMNaCH₃CO₂ pH 4.0 as the sample buffer. The column was washed with 31.5 mlof sample buffer. The enzyme was eluted with a gradient up to 15% (over120 ml) of 1 M NaCH₃CO₂ pH 4.0 and then with a gradient from 15% to 100%(over 90 ml) of 1 M NaCH₃CO₂ pH 4.0. Fractions of 2 ml were collected,assayed for beta-glucosidase activity, and fractions 93 to 107 werepooled based on specific activity and purity (SDS-PAGE).

The retentate (2 ml) from the Source S step was loaded onto a Superdex200 H10/300 GL column (Amersham Bioscience, Uppsala, Sweden) using 100mM NaCH₃CO₂ pH 4.8 with 200 mM NaCl as the sample buffer. The enzyme waseluted with 50 ml of the same buffer. Fractions of 0.5 ml werecollected, assayed for beta-glucosidase activity, and fractions 28 to 31were pooled based on specific activity and purity (SDS-PAGE).

The retentate (8.0 ml) from the Superdex 200 step was loaded onto a 1 mlPhenyl Sepharose HP column (Amersham Bioscience, Uppsala, Sweden) using1 M (NH₄)₂SO₄, 50 mM NaCH₃CO₂ pH 4.8 as the sample buffer. The columnwas washed with 17.0 ml of the sample buffer. The enzyme was eluted witha gradient up to 100% (over 70 ml) of 50 mM NaCH₃CO₂ pH 4.8. Fractionsof 0.5 ml were collected, assayed for beta-glucosidase activity, andfractions 73 to 78 were pooled based on specific activity and purity(SDS-PAGE).

SDS-PAGE of the purified beta-glucosidase showed only one band atapproximately 115 kDa. Isoelectric focusing was performed with aPharmacia PhastSystem using IEF gels, pH 3-9 and a standard mix with pls3.5-9.3. The gel was stained by the silver method for PhastGel IEFmedia. The isoelectric point was determined to be approximately 3.9.

Example 4 N-terminal Sequencing

A 100 μl aliquot of purified Penicillium brasilianum beta-glucosidase(Example 3) was added to 100 μl of SDS-PAGE sample buffer (4 ml of 0.5 MTRIS-HCl pH 6.8, 20 ml of 10% SDS, 20 ml of glycerol (87%), 56 ml ofMilli Q filtered H₂O, and 15 grains of bromphenol blue) in an Eppendorftube and heated to 95° C. for 4 minutes. Following heating four 20 μlaliquots of the diluted sample were applied separately to a precast4-20% SDS polyacrylamide gel (Invitrogen, Carlsbad, Calif., USA). Inaddition to the four lanes containing the sample, a Mark 12 proteinstandard mixture.

The gel was run in an Xcell SureLock™ gel apparatus for 90 minutes withinitial power settings of 40 mA at maximum 135 V. Followingelectrophoresis the gel was incubated for 5 minutes in a blottingsolution consisting of 10 mM CAPS pH 11 containing 6% methanol. AProBlott membrane (Applied Biosystems, Foster City, Calif., USA) waswetted for 1 minute in pure methanol before being placed in the blottingsolution for 5 minutes in order to saturate the membrane with 10 mM CAPSpH 11 containing 6% methanol.

Electroblotting was carried out in a Semi Dry Blotter II apparatus(KemEnTec, Copenhagen, Denmark) as follows. Six pieces of Whatman no. 1paper wetted in the blotting solution were placed on the positiveelectrode of the blotting apparatus followed by the ProBlott membrane,the polyacrylamide gel, and six pieces of Whatman no. 1 paper wetted inblotting solution. The blotting apparatus was assembled thereby puttingthe negative electrode in contact with the upper stack of Whatman no. 1paper. A weight of 11.3 kg was placed on top of the blotting apparatus.The electroblotting was performed at a current of 175 mA for 180minutes.

Following the electroblotting the ProBlott membrane was stained for 1minute in 0.1% (w/v) Coomassie Brilliant Blue R-250 dissolved in 60%methanol, 1% acetic acid, 39% H₂O. Destaining of the ProBlott membranewas performed in 40% aqueous methanol for 5 minutes before the membraneswere rinsed in deionized water. Finally the ProBlott membrane wasair-dried.

For N-terminal amino acid sequencing two pieces of the ProBlott membraneconsisting of a 115 kDa band were cut out and placed in the blottingcartridge of an Applied Biosystems Procise Protein Sequencer (AppliedBiosystems, Foster City, Calif., USA). The N-terminal sequencing wascarried out using the method run file for PVDF membrane samples (Pulsedliquid PVDF) according to the manufacturer's instructions.

The N-terminal amino acid sequence was deduced from the resultingchromatograms by comparing the retention time of the peaks in thechromatograms to the retention times of the PTH-amino-acids in thestandard chromatogram.

The N-terminal amino acid sequence of the purified Penicilliumbrasilianum beta-glucosidase was determined directly using a Procise 494HT Sequencing System (Applied Biosystems, Foster City, Calif., USA). TheN-terminal sequence was determined to beAla-Ile-Glu-Ser-Phe-Ser-Glu-Pro-Phe-Tyr-Pro-Ser-X-X-Met-Asn (amino acids37 to 52 of SEQ ID NO: 2). X defines an undetermined amino acid residue.

Example 5 PCR Amplifications

Based on the N-terminal amino acid sequence of the purified Penicilliumbrasilianum beta-glucosidase (Example 4), a forward primer was designedas shown below using the CODEHOP strategy (Rose et al., 1998, NucleicAcids Res. 26: 1628-35). From database information on otherbeta-glucosidases, a reverse primer was designed as shown below usingthe CODEHOP strategy.

Forward Primer:

-   5′-GCGCTATCGAGTCTTTCTCTGARCCNTTYTA-3′ (SEQ ID NO: 3)    Reverse Primer:-   5′-GTCGGTCATGACGAAGCCNKGRAANCC-3′ (SEQ ID NO: 4)    where R=A or G, Y=C or T, K=G or T and N=A, C, G or T

Amplification reactions (30 μl) were prepared using approximately 1 μgof Penicillium brasilianum genomic DNA as template. In addition, eachreaction contained the following components: 30 pmol of the forwardprimer, 30 pmol of the reverse primer, 200 μM each of dATP, dCTP, dGTP,and dTTP, 1× AmpliTaq polymerase buffer (Applied Biosystems, FosterCity, Calif., USA), and 0.5 unit of AmpliTaq polymerase (5.0 U/μl,Applied Biosystems, Foster City, Calif., USA). The reactions wereincubated in a Robocycler (Stratagene, La Jolla, Calif., USA) programmedfor 1 cycle at 96° C. for 3 minutes and at 72° C. for 3 minutes; 34cycles each at 95° C. for 0.5 minute, 56° C. for 0.5 minutes, and 72° C.for 1.5 minutes; 1 cycle at 72° C. for 7 minutes; and a soak cycle at 6°C. Taq polymerase was added at 72° C. in the first cycle.

PCR reaction products were separated on a 2% agarose gel (Amresco,Solon, Ohio, USA) using TAE buffer. A band of approximately 840 bp wasexcised from the gel and purified using a MiniElute™ Gel Extraction Kit(QIAGEN Inc., Valencia, Calif., USA) according to the manufacturer'sinstructions. The purified PCR product was subsequently cloned into apCR2.1 TOPO vector (Invitrogen, Carlsbad, Calif., USA) according to themanufacturer's instructions to produce a vector designated pCR2.1 GH3A(FIG. 2) and analyzed by DNA sequencing to confirm its identity as aFamily 3 glycosyl hydrolase.

Example 6 Screening of Genomic Library

Colony lifts were performed (Maniatis et al., 1982, Molecular Cloning, ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.)and the DNA was cross-linked onto Hybond N+ membranes (Amersham,Arlington Heights, Ill.) for 2 hours at 80° C. The membranes from thecolony lifts were pre-wetted using 0.2×SSC (0.03 M NaCl, 0.003 M sodiumcitrate), 0.2% SDS. The pre-wetted filters were placed in a beaker with7.5 ml of hybridization solution (6×SSPE [0.9 M NaCl, 0.06 M NaH₂PO₄,and 6 mM EDTA], 7% SDS) per filter at 68° C. in a shaking water bath for0.5 hour. The subcloned product of the PCR amplification described inExample 5 was amplified from pCR2.1 GH3A by PCR amplification usingprimers homologous to the vector, as shown below.

-   5′-CTTGGTACCGAGCTCGGATCCACTA-3′ (SEQ ID NO: 5)-   5′-ATAGGGCGAATTGGGCCCTCTAGAT-3′ (SEQ ID NO: 6)

Amplification reactions (30 μl) were prepared using approximately 50 ngof pCR2.1 GH3A as template. In addition, each reaction contained thefollowing components: Fifty picomoles of each of the primers, 1× Taqbuffer (New England Biolabs, Beverly, Mass.), 15 pmol each of dATP,dTTP, dGTP, and dCTP, and 0.5 units of Taq DNA polymerase (New EnglandBiolabs, Beverly, Mass., USA). The reactions were incubated in aRobocycler programmed for 1 cycle at 94° C. for 1 minute; and 20 cycleseach at 94° C. for 30 seconds, 55° C. for 60 seconds, and 72° C. for 1minute. The heat block then went to a 4° C. soak cycle. The reactionproducts were isolated on a 2.0% agarose gel using TAE buffer, and a 1kb product band was excised from the gel and purified using a QIAquickGel Extraction Kit (QIAGEN Inc., Valencia, Calif., USA) according to themanufacturer's instructions.

Approximately 40 ng was random-primer labeled using a StratagenePrime-It II Kit (Stratagene, La Jolla, Calif., USA) according to themanufacturer's instructions. The radiolabeled gene fragment wasseparated from unincorporated nucleotide using a MinElute PCRPurification Kit (QIAGEN Inc., Valencia, Calif., USA).

The radioactive probe was denatured by adding 5.0 M NaOH to a finalconcentration of 0.5 M, and added to the hybridization solution at anactivity of approximately 0.5×10⁶ cpm per ml of hybridization solution.The mixture was incubated for 10 hours at 68° C. in a shaking waterbath. Following incubation, the membranes were washed three times in0.2×SSC, 0.2% SDS at 68° C. The membranes were then dried on blottingpaper for 15 minutes, wrapped in SaranWrap™, and exposed to X-ray filmovernight at −80° C. with intensifying screens (Kodak, Rochester, N.Y.,USA).

Colonies producing hybridization signals with the probe were inoculatedinto 1 ml of LB medium supplemented with 100 μg of ampicillin per ml andcultivated overnight at 37° C. Dilutions of each solution were made and100 μl were plated onto LB agar plates supplemented with 100 μg ofampicillin per ml. The dilution for each positive that produced about 40colonies per plate was chosen for secondary lifts. The lifts wereprepared, hybridized, and probed as above. Two colonies from eachpositive plate were inoculated into 3 ml of LB medium supplemented with100 μg of ampicillin per ml and cultivated overnight at 37° C.

Miniprep DNA was prepared from each colony using a Bio Robot 9600(QIAGEN Inc, Valencia, Calif., USA) according to the manufacturer'sprotocol. The size of each insert was determined by Eco RI digestion andagarose gel electrophoresis. Two clones designated AB1 and AB2 eachcontained an approximately 4.5 kb insert. Sequencing revealed that theclones were identical, and they were hereafter referred to as pKKAB(FIG. 3).

Example 7 Characterization of the Penicillium brasilianum GenomicSequence Encoding Beta-Glucosidase

DNA sequencing of the Penicillium brasilianum beta-glucosidase gene frompKKAB was performed with an Applied Biosystems Model 3700 Automated DNASequencer (Applied Biosystems, Foster City, Calif., USA) using theprimer walking technique with dye-terminator chemistry (Giesecke et al.,1992, J. Virol. Methods 38: 47-60).

The genomic coding sequence (SEQ ID NO: 1) and deduced amino acidsequence (SEQ ID NO: 2) are shown in FIGS. 1A and 1B. The genomic codingsequence of 2751 bp (including stop codon) encodes a polypeptide of 878amino acids with a calculated molecular mass of 96,725 Da, interruptedby 2 introns of 57 bp (85-141 bp) and 57 bp (312-368 bp). The % G+Ccontent of the gene is 51.9% and of the mature protein coding region(nucleotides 171 to 2753 of SEQ ID NO: 1) is 52%. Using the SignalPsoftware program (Nielsen et al., 1997, Protein Engineering 10:1-6), asignal peptide of 19 residues was predicted. Based on the N-terminalsequence of the beta-glucosidase, residues 20 through 36 appear toconstitute a propeptide region that is proteolytically cleaved duringmaturation. The predicted mature protein contains 842 amino acids.

A search for similar sequences in public databases was carried out withthe FASTA program package, version 3.4 (Pearson and D. J. Lipman, 1988,PNAS 85:2444, and Pearson, 1990, Methods in Enzymology 183:63) usingdefault parameters. The pairwise alignments from the package'sSmith-Waterman algorithm (Waterman et al., 1976, Adv. Math. 20: 367)were used for determination of percent identity. Default parametersincluded a gap open penalty of −12, a gap extension penalty of −2, andthe BLOSUM50 comparison matrix. The alignments showed that the deducedamino acid sequence of the Penicillium brasilianum gene encoding a GH3Apolypeptide having beta-glucosidase activity shared 63.8% identity(including gaps) to the deduced amino acid sequence of a hypotheticalprotein from Neurospora crassa (accession number Q7RWP2) and 61.8%identity to a characterized glycosyl hydrolase Family 3 beta-glucosidasefrom Aspergillus cellulolyticus (accession number ABB07868).

E. coli TOP10 cells (Invitrogen, Carlsbad, Calif., USA) containingplasmid pKKAB were deposited with the Agricultural Research ServicePatent Culture Collection, Northern Regional Research Center, 1815University Street, Peoria, Ill., 61604, as NRRL B-30860, with a depositdate of Jul. 8, 2005.

Example 8 Construction of an Aspergillus oryzae Beta-glucosidaseExpression Plasmid

The Aspergillus expression plasmid pJaL721 (WO 03/008575) consists of anexpression cassette based on the Aspergillus niger neutral amylase IIpromoter fused to the Aspergillus nidulans triose phosphate isomerasenon-translated leader sequence (NA2/tpi) and the Aspergillus nigeramyloglucosidase terminator (Tamg). Also present on the plasmid is theselective marker amdS from Aspergillus nidulans enabling growth onacetamide as sole nitrogen source and the URA3 marker from Saccharomycescerevisiae enabling growth of the pyrF defective Escherichia coli strainDB6507 (ATCC 35673). Transformation into E. coli DB6507 was performedusing the Saccharomyces cerevisiae URA3 gene as selective marker asdescribed below.

E. coli DB6507 was made competent by the method of Mandel and Higa,1970, J. Mol. Biol. 45: 154. Transformants were selected on solid M9medium (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, MolecularCloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.)supplemented per liter with 1 g of casamino acids, 500 μg of thiamine,and 10 mg of kanamycin.

The beta-glucosidase gene was cloned into pJaL721 as described below.The beta-glucosidase gene from Penicillium brasilianum was amplified byPCR using the following two oligonucleotide primers:

Forward PCR: (SEQ ID NO: 7)5′-AATTTGATCACACCATGCAGGGTTCTACAATCTTTCTGCC-3′ Reverse PCR:(SEQ ID NO: 8) 5′-TTAACTCGAGTTACTCCAATTGTGAGCTCAGCGG-3′

To facilitate cloning a restriction enzyme site was inserted into the 5′end of each primer where the forward primer contained a Bcl I site andthe reverse primer contained an Xho I site.

The AB clone (Example 6) was used as template in the PCR reaction. Thereaction was performed in a volume of 50 μl containing 1.0 unit ofPhusion (Finnzymes Oy, Espoo, Finland), 1× Phusion buffer HF (FinnzymesOy, Espoo, Finland), 25 ng of clone AB, 250 μM of each dNTP, and 50 pmolof each of the two primers described above. The amplification wascarried out in a PTC-220 DNA Engine Dyad Peltier Thermal Cycler (MJResearch, Inc., Waltham, Mass., USA) programmed for 1 cycle at 95° C.for 5 minutes; 24 cycles each at 94° C. for 0.5 minute, 58° C. for 0.5minute, and 68° C. for 4.0 minutes; and 1 cycle at 68° C. for 15minutes. The hot start PCR technique (Chou et al., 1992, Nucleic AcidsRes. 20: 1717) was used and the Phusion polymerase was added after 1minute of the first cycle.

The PCR reaction produced a single DNA fragment of approximately 2700 bpin length. The fragment was digested with Bcl I and Xho I and isolatedby agarose gel electrophoresis, purified, and cloned into pJaL721digested with Bam HI and Xho I, resulting in a plasmid designated pKBK01(FIG. 4). The sequence of the beta-glucosidase gene in pKBK01 wasverified by DNA sequencing.

Example 9 Expression of the Penicillium brasilianum Beta-glucosidase inAspergillus oryzae

Aspergillus oryzae BECh2 (WO 00/30322) was transformed with 5 μg ofpKBK01 as described by Christensen et al., 1988, Biotechnology 6:1419-1422.

Transformants were cultivated in 50 ml tubes for 4 days at 30° C. in 10ml of YPM medium. The whole broths were centrifuged at 12,100×g and thesupernatants removed. The supernatants were analyzed by SDS-PAGE using aCriterion XT Precast Gel, 10% Bis-Tris gel in a XT MES buffer (BioRadLaboratories, Hercules, Calif., USA) according to the manufacturer'sinstructions. A 10 μl volume of supernatant was mixed with 9 μl ofsample buffer (0.125 M Tris-HCl pH 6.8, 20% glycerol, and 4.6% SDS) and1 μl of 1 M dithiothreitol, and heated to 96° C. for 5 minutes. In 8 outof 28 supernatants, one band of approximately 115 kDa was visible in therange of the standards 35 kDa to 150 kDa by SDS-PAGE. The supernatantsresulting in a band at approximately 115 kDa also containedbeta-glucosidase activity, assayed as described in Example 3. The higherthe intensity of the band, the higher beta-glucosidase activity measuredin the same supernatant.

One transformant was designated Aspergillus oryzae KBK01.

Example 10 Production and Purification of Recombinant Penicilliumbrasilianum Beta-glucosidase

Aspergillus oryzae transformant KBK01 was grown in a bioreactor for 24hours in a medium composed per liter of 60 g of sucrose, 10 g ofMgSO₄.H₂0, 10 g of KH₂PO₄, 15 g of K₂SO₄, 20 g of citric acid, 50 g ofyeast extract, 0.5 ml of trace metals, and 1 ml of pluronic acid. Thetrace metals was composed per liter of 14.28 g of ZnSO₄.7H₂O, 2.50 g ofCuSO₄.5H₂O, 2.5 g of NiCl₂.6H₂O, 13.8 g of FeSO₄.7H₂O, 8.5 g ofMnSO₄.H₂O, and 3.0 g of citric acid. After 1 day, a maltose solution wasfed into the bioreactor composed per liter of 350 g of 75% maltosesolution, 5 g of citric acid, 10 g of yeast extract, 0.5 ml of tracemetals, and 5 ml of pluronic acid. After 5 days the cultivation wasstopped.

The biomass was removed from 2.5 liters of fermentation broth bycentrifugation and filtration. The resulting supernatant was brought to5 liters with deionized water and ultrafiltrated on a Filtron with anOS10C72 10 kDa membrane (Filtron, USA). The resulting volume of 1.2liters was adjusted to pH 8.5.

Beta-glucosidase activity was measured as described in Example 3.Protein concentration was determined as described in Example 3. SDS-PAGEanalysis was performed as described in Example 3. Elution of thebeta-glucosidase was monitored at 280 nm.

The beta-glucosidase solution was loaded onto a Q-Sepharose Fast Flowcolumn (Amersham Biosciences, Uppsala, Sweden) pre-equilibrated with 25mM Tris pH 8.5. The beta-glucosidase was eluted with a 0 to 1 M NaClgradient (5 column volumes) in 25 mM Tris pH 8.5. Fractions containingthe beta-glucosidase were pooled in a volume of 105 ml.

A portion of the pool (40 ml) from the Q-Sepharose step was furtherpurified on a Sephacryl S-200 column pre-equilibrated in 0.1 M sodiumacetate pH 6.0. The beta-glucosidase was eluted with the same buffer ina volume of 68 ml.

The protein content was determined from the absorbance at 280 nm and theextinction coefficient calculated from the primary structure of thebeta-glucosidase.

The purification was followed by SDS-PAGE. The samples were boiled for 2minutes with an equal volume of 2× sample buffer and ⅕ volume of 1% PMSFand loaded onto a 4-20% Tris-glycine gel from Novex. The gel was stainedwith GelCode Blue Stain Reagent and destained with water. SDS-PAGErevealed one band of approximately 115 kDa.

Example 11 Characterization of Purified Recombinant Penicilliumbrasilianum Beta-glucosidase

The purified recombinant Penicillium brasilianum beta-glucosidasedescribed in Example 10 was characterized with regard to pH optimum,temperature optimum, temperature stability, and substrate specificity.

pH optimum and tTemperature optimum. The beta-glucosidase activity wasmeasured at temperatures from 20° C. to 90° C. and at pH values of 3.0to 8.0. The purified beta-glucosidase was diluted in MilliQ water toensure that the 4-nitrophenol developed in the assay was within thestandard curve. The substrate was 1 mM4-nitrophenyl-beta-D-glucopyranoside in 50 mM sodium citrate adjusted topH 3.18, 4.16, 4.86, 6.17 and in 50 mM sodium carbonate adjusted to pH7.07 and 8.13. The activity was measured for 10 minutes and the reactionwas terminated with 0.5 M glycine/NaOH pH 10 with 2 mM EDTA.

FIG. 5 shows the relative activity of the Penicillium brasilianum strainIBT 20888 beta-glucosidase at different pH values as a function oftemperature.

FIG. 6 shows the relative activity of the Penicillium brasilianum strainIBT 20888 beta-glucosidase at different temperatures as a function ofpH.

Temperature stability for Novozym 188 and the Penicillium brasilianumstrain IBT 20888 beta-glucosidase. The stability of the Penicilliumbrasilianum beta-glucosidase and Novozym 188 (Novozymes A/S, Bagsværd,Denmark) were tested at temperatures from 20° C. to 67.5° C. and at pHvalues of 3 to 8 over a period of 24 hours. The enzyme preparations werediluted 2000-fold in the incubation buffer. Incubation buffers of pH 3to pH 6 contained 10 mM sodium citrate adjusted to the desired pH andincubation buffers of pH 7 and pH 8 contained 10 mM sodium carbonateadjusted to the desired pH. The residual activity was measured at roomtemperature during a period of 10 minutes. The substrate was 1 mM4-nitrophenyl-beta-D-glucopyranoside in 50 mM sodium citrate adjusted topH 4.80, and the reaction was terminated with 0.5 M glycine/NaOH pH 10containing 2 mM EDTA.

FIG. 7 shows the residual activity of Novozym 188 after 24 hours ofincubation at different temperature and pH (n=2).

FIG. 8 shows the residual activity of the Penicillium brasilianum strainIBT 20888 beta-glucosidase after 24 hours of incubation at differenttemperature and pH (n=3)

The beta-glucosidase from Penicillium brasilianum strain IBT 20888 wasstable at pH 4 to pH 6 up to 60° C. for a period of 24 hours. Novozym188 was stable at pH 4 and pH 5 up to 50° C. for a period of 24 hoursand at pH 6 up to 40° C. for a period of 24 hours.

Kinetic parameters for the Penicillium brasilianum strain IBT 20888beta-glucosidase. Substrate 4-nitrophenyl-beta-D-glucopyranoside.Kinetic parameters were measured using4-nitrophenyl-beta-D-glucopyranoside at concentrations between 0.07 and2 mM in 50 mM sodium citrate pH 4.80. The activity was measured for 2minutes at room temperature and the reaction was terminated with 0.5 Mglycine/NaOH pH 10 with 2 mM EDTA, and measured as described in Example3.

The Michaelis-Menten constant k_(m) and the maximum reaction rate weredetermined from four independent dilutions of the enzyme. Themeasurements showing substrate inhibition were omitted in thedetermination of the kinetic parameters. The parameters were determinedfrom a Lineweaver-Burk plot to be k_(m)=0.077±0.021 mM andV_(max)=78.2±7.2 U/mg enzyme. Using a Hanes plot to determine theparameters resulted in a deviation of the parameters of less than onepercent.

FIG. 9 shows the initial reaction rate at different4-nitrophenyl-beta-D-glucopyranose concentrations for the Penicilliumbrasilianum strain IBT 20888 beta-glucosidase.

Substrate cellobiose. Kinetic parameters were measured using cellobioseat concentrations between 0.08 and 10 mM in 50 mM sodium acetate pH4.80. The activity was measured for 5 minutes at room temperature andthe reaction was terminated with 0.5 M glycine/NaOH pH 10 with 2 mMEDTA, and then heated to 65° C. for 10 minutes. The pH was then adjustedto pH 7.1 with 1 M HCl and measured with Ecoline S+Glucose (DiaSysDiagnostics Systems GmbH, Holzheim, Germany). The Michaelis-Mentenparameters were determined with a non-linear curve fitter using theMarquardt-Levenberg algorithm (SigmaPlot 9.01, Systat Software, Inc.).

The Michaelis-Menten constant k_(m) and the maximum reaction rate weredetermined for the hydrolysis of cellobiose to be 1.58 mM and 28 U/mg,respectively. One unit is defined as the amount of enzyme hydrolyzing 1μmole of cellobiose per minute.

FIG. 10 shows the initial reaction rate at different cellobioseconcentrations for the Penicillium brasilianum strain IBT 20888beta-glucosidase.

Deposit of Biological Material

The following biological material has been deposited under the terms ofthe Budapest Treaty with the Agricultural Research Service PatentCulture Collection, Northern Regional Research Center, 1815 UniversityStreet, Peoria, Ill., 61604, and given the following accession number:

Deposit Accession Number Date of Deposit E. coli TOP10 pKKAB NRRLB-30860 Jul. 8, 2005

The strain has been deposited under conditions that assure that accessto the culture will be available during the pendency of this patentapplication to one determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. §1.14 and 35 U.S.C.§122. The deposit represents a substantially pure culture of thedeposited strain. The deposit is available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

The invention described and claimed herein is not to be limited in scopeby the specific aspects herein disclosed, since these aspects areintended as illustrations of several aspects of the invention. Anyequivalent aspects are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. In the case ofconflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which areincorporated by reference in their entireties.

1. An isolated polynucleotide that encodes for a signal peptidecomprising or consisting of amino acids 1 to 19 of SEQ ID NO: 2, apropeptide comprising or consisting of amino acids 20 to 36 of SEQ IDNO: 2, or a prepropeptide comprising or consisting of amino acids 1 to36 of SEQ ID NO:
 2. 2. The isolated polynucleotide of claim 1 thatencodes for a signal peptide comprising or consisting of amino acids 1to 19 of SEQ ID NO:
 2. 3. The isolated polynucleotide of claim 1 thatencodes for a propeptide comprising or consisting of amino acids 20 to36 of SEQ ID NO:
 2. 4. The isolated polynucleotide of claim 1 thatencodes for a prepropeptide comprising or consisting of amino acids 1 to36 of SEQ ID NO:
 2. 5. A nucleic acid construct comprising a codingsequence operably linked to the polynucleotide of claim 1, wherein thecoding sequence is foreign to the polynucleotide that encodes for thesignal peptide, the propeptide, or the prepropeptide.
 6. The nucleicacid construct of claim 5, wherein the coding sequence is operablylinked to a polynucleotide that encodes for a signal peptide comprisingor consisting of amino acids 1 to 19 of SEQ ID NO: 2, and wherein thecoding sequence is foreign to the polynucleotide that encodes for thesignal peptide.
 7. The nucleic acid construct of claim 5, wherein thecoding sequence is operably linked to a polynucleotide that encodes fora signal peptide consisting of amino acids 1 to 19 of SEQ ID NO: 2, andwherein the coding sequence is foreign to the polynucleotide thatencodes for the signal peptide.
 8. The nucleic acid construct of claim5, wherein the coding sequence is operably linked to a polynucleotidethat encodes for a propeptide comprising or consisting of amino acids 20to 36 of SEQ ID NO: 2, and wherein the coding sequence is foreign to thepolynucleotide that encodes for the propeptide.
 9. The nucleic acidconstruct of claim 5, wherein the coding sequence is operably linked toa polynucleotide that encodes for a propeptide consisting of amino acids20 to 36 of SEQ ID NO: 2, and wherein the coding sequence is foreign tothe polynucleotide that encodes for the propeptide.
 10. The nucleic acidconstruct of claim 5, wherein the coding sequence is operably linked toa polynucleotide that encodes for a prepropeptide comprising orconsisting of amino acids 1 to 36 of SEQ ID NO: 2, and wherein thecoding sequence is foreign to the polynucleotide that encodes for theprepropeptide.
 11. The nucleic acid construct of claim 5, wherein thecoding sequence is operably linked to a polynucleotide that encodes fora prepropeptide consisting of amino acids 1 to 36 of SEQ ID NO: 2, andwherein the coding sequence is foreign to the polynucleotide thatencodes for the prepropeptide.
 12. A recombinant host cell comprising acoding sequence operably to the polynucleotide of claim 1, wherein thecoding sequence is foreign to the polynucleotide that encodes for thesignal peptide, the propeptide, or the prepropeptide.
 13. Therecombinant host cell of claim 12, wherein the coding sequence isoperably linked to a polynucleotide that encodes for a signal peptidecomprising or consisting of amino acids 1 to 19 of SEQ ID NO: 2, andwherein the coding sequence is foreign to the polynucleotide thatencodes for the signal peptide.
 14. The recombinant host cell of claim12, wherein the coding sequence is operably linked to a polynucleotidethat encodes for a propeptide comprising or consisting of amino acids 20to 36 of SEQ ID NO: 2, and wherein the coding sequence is foreign to thepolynucleotide that encodes for the propeptide.
 15. The recombinant hostcell of claim 12, wherein the coding sequence is operably linked to apolynucleotide that encodes for a prepropeptide comprising or consistingof amino acids 1 to 36 of SEQ ID NO: 2, and wherein the coding sequenceis foreign to the polynucleotide that encodes for the prepropeptide. 16.A method of producing a protein, comprising (a) cultivating arecombinant host comprising a coding sequence that encodes for theprotein operably to the polynucleotide of claim 1, wherein the codingsequence is foreign to the polynucleotide that encodes for the signalpeptide, the propeptide, or the prepropeptide; and (b) recovering theprotein.
 17. The method of claim 16, wherein the coding sequence isoperably linked to a polynucleotide that encodes for a signal peptidecomprising or consisting of amino acids 1 to 19 of SEQ ID NO: 2, andwherein the coding sequence is foreign to the polynucleotide thatencodes for the signal peptide.
 18. The method of claim 16, wherein thecoding sequence is operably linked to a polynucleotide that encodes fora propeptide comprising or consisting of amino acids 20 to 36 of SEQ IDNO: 2, and wherein the coding sequence is foreign to the polynucleotidethat encodes for the propeptide.
 19. The method of claim 16, wherein thecoding sequence is operably linked to a polynucleotide that encodes fora prepropeptide comprising or consisting of amino acids 1 to 36 of SEQID NO: 2, and wherein the coding sequence is foreign to thepolynucleotide that encodes for the prepropeptide.